WO2006109424A1 - Dnas encoding protein having function of forming and regulating cellulosic fiber cell wall in tree trunk and promoter dnas of the same - Google Patents

Dnas encoding protein having function of forming and regulating cellulosic fiber cell wall in tree trunk and promoter dnas of the same Download PDF

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Publication number
WO2006109424A1
WO2006109424A1 PCT/JP2006/305402 JP2006305402W WO2006109424A1 WO 2006109424 A1 WO2006109424 A1 WO 2006109424A1 JP 2006305402 W JP2006305402 W JP 2006305402W WO 2006109424 A1 WO2006109424 A1 WO 2006109424A1
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dna
seq
trunk
protein
amino acid
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PCT/JP2006/305402
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French (fr)
Japanese (ja)
Inventor
Shigeru Sato
Nanae Yamada
Shiho Nakamoto
Takashi Hibino
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Oji Paper Co., Ltd.
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Priority to JP2007512437A priority Critical patent/JPWO2006109424A1/en
Publication of WO2006109424A1 publication Critical patent/WO2006109424A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the present invention relates to DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk, a promoter DNA thereof, and use thereof.
  • the present invention also relates to DNA encoding plant transcription factors, promoter DNA fragments, and uses thereof.
  • wood fibers Most of the woody biomass in the trunk is composed of tissue cells called wood fibers.
  • the specific gravity of the wood fiber structure, cell wall structure and components (cellulose, hemicellulose, lignin), etc. are the main factors that determine the properties of wood fibers.
  • Non-Patent Document 1 Non-Patent Document 2
  • the fluctuation phenomenon due to the ground height is the vertical fluctuation of the material in the trunk
  • the fluctuation phenomenon by the species Is known as inter-species variation.
  • cellulose increases in cell wall components, while lignin and hemicellulose decrease.
  • Non-Patent Documents 1 to 3 Although details are unknown, it is thought that the difference in the properties of the wood fibers formed is caused by the difference in cell wall synthesis in each wood fiber-forming tissue. The gene groups responsible for cell wall synthesis with such different properties have not been identified. [0007] In recent years, gene analysis studies at the genome level using model plants such as herbs have been actively conducted. In herbs, only a few wood fibers are formed in specific organs at specific times.
  • Non-patent Document 4 Fibrous fiber cells differentiate from the formation layer tissue and are called wood fibers, whereas herbaceous fiber cells mainly divide the parenchyma force between the vascular bundles, and the inter-bundle fibers ( It is called interfascicular fiber).
  • wood fibers Fibrous fiber cells differentiate from the formation layer tissue and are called wood fibers
  • herbaceous fiber cells mainly divide the parenchyma force between the vascular bundles, and the inter-bundle fibers ( It is called interfascicular fiber).
  • interfascicular fiber the interfascicular fiber
  • Plant fiber formation-related genes and their regulatory factors cannot be distinguished and identified simply by homology search or motif search with known regulatory factors.
  • many enzymes are involved in the synthesis pathway of cellulose and lignin, which are the main components of wood fiber cell walls, and these genes form a large family in the plant genome (Non-patent Document 5).
  • Non-patent Document 5 Furthermore, within the family 1, there are those related to the synthesis of the primary cell wall and those related to the synthesis of the secondary cell wall, and the functioning places and roles are divided in various ways. Sequence motifs and homology search capabilities simply indicate high homology and cannot identify roles. For example, about 40 cellulose synthases (CesA) involved in cellulose synthesis are expected in Arabidopsis thaliana!
  • RSW1 Non-patent document 6
  • Ces A essential for cellulose synthesis of the primary wall
  • IRX3 Non-patent document 7
  • Ce sA essential for the synthesis of secondary wall cellulose
  • a homologous search factor NtLIMl identified in tobacco as a regulator of the biopropanoid biosynthesis system represented by lignin contained in wood fiber is known (Searching at http://www.ncbi.nlm.nih.gov/blast/) shows high homology with many LIM proteins. Even rat LI M proteins that do not form wood fibers are hit with a high homology score. In Arabidopsis, at least 10 LIM proteins and a high score (56-87%) are hit, but it is unclear which LIM protein is involved in the control of the lignin synthesis system in Arabidopsis. Thus, since the role of a regulatory factor cannot be identified only by homology with known regulatory factors, detailed expression analysis and the like must be performed. However, there is no knowledge of comprehensive expression analysis of regulatory factors in Kashiwagi.
  • a variety of wood fiber cell wall synthesis genes are expected to exist in woody plants, which are based only on the difference in the genes that act during fiber formation between grass and wood. For example, because the morphology of leaves and trunks is different between young and middle age, it is expected that multiple tree fiber cell wall synthetic genes that function according to age will function. Also, as mentioned above, vertical fluctuations due to drought height and fluctuations among varieties due to varieties occur, so that a plurality of tree fiber cell wall synthetic genes that are different within the same individual or between species function uniquely. ! I think. These tree fiber cell wall synthetic genes only show high homology with each other in sequence, and it is not possible to distinguish which belongs to which level of wood fiber cell wall synthetic genes. In order to make this distinction, detailed expression analysis at the wood fiber formation site is necessary. However, there is no such knowledge.
  • Non-Patent Documents 7 and 9 Also in woody plants, research on the analysis of silkworms at the genome level using poplar and pine has been conducted (Non-Patent Documents 7 and 9). However, the materials used are separate organs and tissue units such as roots and leaves' formation layers, and only report gene expression information in them. There is no comprehensive knowledge at the genome level regarding gene groups involved in various levels of wood fiber cell wall synthesis, their promoter information, and expression information by conducting expression analysis in different types of tree fiber-forming tissues on the ground.
  • the growth state of the afforestation tree is judged by appearance, and in order to know the quality as a pulp material, a trunk that is a pulp material by a chemical method after cutting is used.
  • Wood fiber analysis material analysis
  • As for the growth state it relies on experience and intuition, so it cannot grasp problems such as lack of nutrients that cannot be distinguished by appearance. And since the material is only divided after logging, what is the power of tree fiber formation (material) in planted trees during growth, what kind of tree fiber will be formed in the future? There is no policy of working (fertilization, thinning, etc.) to produce good materials.
  • trunk fiber formation material during growth is very important in consideration of plantation operations, selective breeding, and securing high-quality papermaking raw materials.
  • Patent Document 1 Patent No. 3444191
  • Non-Patent Document 1 Ken Shimaji ⁇ Akuji Sudo ⁇ Hirada Hiroshi "Wood Organization", Morikita Publishing, 1976, 111-21 Five
  • Non-Patent Document 2 Satoshi Furuno 'Osamu Sawabe's "Wood Science Course 2 Organization and Material", Kaiseisha, 1994, 109 -137
  • Non-Patent Document 3 Wood Science and Technology. 2001; 35: 229-243.
  • Non-Patent Document 4 Plant Physiology, 2001, 126: 477-479.
  • Non-Patent Document 5 Plant Physiology, 2000, 124: 495-498.
  • Non-Patent Document 6 Science 1998, 279: 717-720.
  • Non-Patent Document 7 Plant Cell 1999, 11: 769-780.
  • Non-Patent Document 8 Masami Iwabuchi ⁇ Kazuo Shinozaki “Dynamics of Plant Genomic Function” Syupurin Gaichi 'Fairark Tokyo, 2001, 1-34
  • Non-Patent Document 9 GenomeBiol. 2002; 3 (12): REVIEWS1033.
  • Non-Patent Document 10 Proc Natl Acad Sci U S A 2001 Dec 4; 98 (25): 14732-7.
  • woody biomass varies depending on the fineness and shape of wood fibers. By artificially controlling the formation of fine fibers and shapes of the wood fibers, woody biomass can be produced as an industrial raw material effectively and efficiently. In addition, the world still depends on fossil resources such as large quantities of oil and natural gas. Now, in order to change this situation with new technology, efficient utilization of trees as woody biomass is the key to power. In overseas countries, the establishment of technology for effective utilization of Kashiwagi biomass is positioned as an important issue at the beginning of this century. In fact, coniferous pine, broad-leaved poplar in the United States, spruce and poplar in Canada, and poplar in Scandinavia are the target species. In order to contribute to more effective and efficient production of woody biomass on a global scale, it is highly necessary to identify genes that control the formation of wood fiber cells in plants.
  • An object of the present invention is to provide a DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk, a DNA encoding a plant transcription factor, a promoter DNA thereof, a plasmid containing these, and a plasmid thereof Plant cells transformed by To provide microorganisms or plants.
  • Another object of the present invention is to provide a protein having a function of forming a tree fiber cell wall of a plant tree trunk and a DNA encoding a plant transcription factor as a test marker for a plant trunk tree part.
  • the present inventors use the prepared eucalyptus EST database to extract a group of genes involved in cell wall synthesis in a eucalyptus tree fiber-forming tissue by microarray analysis, and to determine the height of the ground. Expression variation was examined. As a result, we identified a cell wall synthetic gene group of Eucalyptus tree fiber-forming tissue whose expression is preferentially varied depending on the difference between the above-ground species. Furthermore, their promoter DNA was obtained. It is considered that the above gene group and promoter DNA can be used to control cell wall synthesis and morphogenesis of wood fiber-forming tissues and to control gene expression specific to wood fiber.
  • the gene group of the present invention has an expression level on the xylem side compared to the phloem side in the trunk base and central trunk of Eucalyptus genus Camaldrensis.
  • the expression level is decreased on the xylem side compared to the phloem side, and in the middle trunk, the xylem is compared with the phloem side.
  • the expression level increased on the xylem side compared to the phloem side, and in the trunk of the Eucalyptus globules, the xylem compared to the phloem side.
  • DNA encoding a protein having a function of forming a eucalyptus trunk tree fiber cell wall obtained by the present invention as well as techniques for comprehensively controlling them and expression information.
  • various characteristics such as high cellulose, low lignin, thick cell walls, thin ones, long fiber lengths, short ones, etc. depending on the characteristics of the new transgenic eucalyptus varieties obtained using these genes.
  • Quantitative and qualitative changes are expected.
  • the present invention relates to DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk, a promoter DNA fragment thereof, and use thereof, which provide the following [1] to [18] It is.
  • DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk according to any one of the following (a) to (e).
  • the expression level of at least one DNA described in the following (a) to (e) is detected, and the phloem side and xylem side are detected.
  • a method for examining the growth state of a stem trunk of a plant comprising a step of comparing the expression levels of the DNA.
  • SEQ ID NO: DNA comprising the nucleotide sequence set forth in any of 1-30 or 32-34
  • a promoter DNA comprising the nucleotide sequence of any of the following (a) to (c).
  • SEQ ID NO: 93- The nucleotide sequence according to any one of LOO
  • a recombinant vector comprising the DNA according to any one of [1] to [3] or [10] to [12]
  • the present inventors examined the transcriptional factors involved in eucalyptus tree fiber cell wall formation by microarray analysis, and the expression variation due to differences in the height and species of the ground. It was. As a result, we identified a group of transcription factors that regulate the expression of Eucalyptus tree fiber cell wall-forming genes, whose expression changes predominantly depending on the height of the ground. Furthermore, their promoter DNA was obtained.
  • the gene group of the present invention has an expression level on the xylem side compared to the phloem side in the trunk base and central trunk of the Eucalyptus genus Camaldrensis.
  • the expression level rises on the xylem side compared to the phloem side, and the phloem side and xylem on the trunk base Genes whose expression level is the same on the side of the eucalyptus genus, and that the expression level on the xylem side is higher than that on the phloem side of the eucalyptus genus Camaldrensis and Eucalyptus globules.
  • the expression (level) information of this gene group can be used for quantitative and qualitative advance prediction of plant growth state, wood fiber formation state, and material.
  • appropriate operations weeding, thinning, etc.
  • DNA encoding eucalyptus transcription factors obtained by the present invention, as well as technology and expression information for overall control of them.
  • these genes are used as one result.
  • High cellulose, low lignin, thick cell walls, thin cells, long fiber lengths, short ones Various quantitative and qualitative changes are expected.
  • it is expected to be used to distinguish the ground height, species, and other materials and growth status based on the expression information.
  • the present invention provides the following [1] to [17] regarding DNA encoding plant transcription factors, promoter DNA fragments thereof, and use thereof.
  • the expression level of at least one DNA described in the following (a) to (e) is detected, and the phloem side and xylem side are detected.
  • a method for examining the growth state of a stem trunk of a plant comprising a step of comparing the expression levels of the DNA.
  • SEQ ID NO: consisting of the base sequence described in any one of 101 to 107 or 109 to 111 DNA that hybridizes with DNA under stringent conditions
  • the expression level of at least one DNA described in the following (a) to (e) is detected, and the phloem side and xylem side are detected.
  • a method for examining the growth state of a stem trunk of a plant comprising a step of comparing the expression levels of the DNA.
  • a promoter DNA comprising the nucleotide sequence of any of the following (a) to (c).
  • a recombinant vector comprising the DNA according to any one of [1] to [3], [10] or [11]
  • the DNA of the present invention can be used as a test marker for trunk stem parts of plants.
  • the DNA of the present invention it is possible to examine the growth state of trunk trunks of trees, the formation state of wood fibers, the quality of norp quality, and the selection of plants having useful traits.
  • the present invention provides a DNA encoding a protein having a function of forming a tree fiber cell wall of a plant trunk (a protein involved in cell mouth synthesis and a protein involved in lignin synthesis) and a promoter DNA.
  • DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk in the present invention can be used for control of plant fiber fiber cell formation.
  • the promoter DNA of the DNA can be used to control expression specific to wood fiber tissue or wood fiber formation time.
  • the expression (level) information of DNA encoding a protein that functions to form the tree fiber cell wall of plant trunks is used for quantitative and qualitative prediction of plant growth, fiber formation, and materials. It is considered possible. Therefore, by using these, artificial modification of tree fiber cell morphogenesis, specific expression of protein's tree fiber cells, expression of plant growth state, state of fiber formation, amount of material • It can be used for qualitative advance prediction. As a result, the effectiveness of pulp production It will be possible to increase efficiency, reduce costs, and produce paper with new characteristics.
  • Controlling wood fiber formation in plants has various important implications in the fields of industry and agriculture. For example, modification of the wood fiber properties of Eucalyptus genus Camaldrensis is significant in terms of improving the fiber properties of fiber raw materials such as pulp by increasing the fiber length. In addition, increasing the cellulose and hemicellulose content of Eucalyptus globulae species increases the yield of pulp and the like, and improves the cooking efficiency, which is significant in terms of economy and profitability.
  • the plant from which the DNA of the present invention is derived is not particularly limited, for example, useful crops (including forage crops) such as cereals, vegetables, fruit berries, fiber raw material plants such as pulp, ornamental plants, etc.
  • useful crops including forage crops
  • examples include plants.
  • the plant such as eucalyptus, pine, acacia, poplar, cedar, cypress, bamboo, yew, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower,
  • Examples include ivy, orange, grape, peach, pear, apple, tomato, Chinese cabbage, cabbage, Japanese radish, carrot, cabbage, cucumber, melon, parsley, orchid, chrysanthemum, lily and saffron.
  • examples of the DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree include the DNAs described in any of (a) to (e) below.
  • stringent hybridization conditions include conditions of 60 ° C, standing at room temperature in an O.lxSSC solution, or an equivalent stringency of no ibrida.
  • An example is the condition. Under such conditions, DNA that hybridizes with DNA having the nucleotide sequence ability described in any one of SEQ ID NOs: 1 to 34 can be isolated.
  • DNA is extracted from a plant, a gene library is constructed, and screening is performed under the same conditions, or the extracted DNA is represented by SEQ ID NOs: 1-34. From the base sequence described in any of the sequences, a contiguous neighboring sequence can be easily obtained by the TAIL-PCR method established by Liu et al. Using an arbitrary 20-mer sequence as a primer.
  • the present invention also provides a DNA encoding a protein having 50% or more homology with a protein having the amino acid sequence ability described in any of SEQ ID NOs: 35 to 68.
  • DNA can be isolated by those skilled in the art by generally known methods. For example, hybridization technology (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR) technology (Saiki, RK. Et al., Science, 1985, 230, 1350. Sa iki, RK. Et al., Science 1988, 239, 487.).
  • the DNA consisting of the base sequence described in any one of SEQ ID NOs: 1-34 or a part thereof as a probe, and the DNA consisting of the base sequence described in any one of SEQ ID NOs: 1-34 It is a common practice for those skilled in the art to isolate a DNA having a high homology with a DNA comprising the base sequence described in any one of SEQ ID NOS: 1 to 34 using an oligonucleotide that specifically hybridizes as a primer. Is to get.
  • a hybridization reaction is preferably performed under stringent conditions.
  • stringent hybridization conditions refer to conditions of 6M urea, 0.4% SDS, 0.5 X SSC or equivalent stringency hybridization conditions. It is expected that DNA with higher homology can be isolated under conditions with higher stringency, for example, 6M urea, 0.4% SDS, and 0.1X SSC.
  • the DNA thus isolated is considered to have high homology with the amino acid sequence encoded by the DNA having the nucleotide sequence described in any one of SEQ ID NOs: 1 to 34 at the amino acid level.
  • High homology means at least 50% or more of the entire amino acid sequence, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more. Refers to the identity of the sequence.
  • score 50
  • wo rdlength 3.
  • the DNA of the present invention includes an amino acid in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 35 to 68.
  • DNA encoding a protein consisting of a sequence is included.
  • Modification of amino acids in a protein is usually within 50 amino acids of all amino acids, preferably within 30 amino acids, more preferably within 10 amino acids, and even more preferably within 3 amino acids.
  • the amino acid modification can be performed using, for example, “Transformer Site—directed Mutagenesis Kit” or “ExSite PCR—Based 3 ⁇ 4ite—directed Mutagenesis KitJ (Clontech), for mutation or substitution, Deletion can be performed using “Quantum le ap Nested Deletion Kit J (Clontech)”.
  • the DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, cDNA, chemically synthesized DNA, and the like.
  • Genomic DNA is, for example, a genomic DNA prepared according to the method described in the literature (Rogers and Bendich, Plant Mol. Biol, 1985, 5, 69.) as a saddle type, and the base sequence of the DNA of the present invention (for example, sequence No .: PCR (Saiki et al. Scienc) using a primer prepared on the basis of the nucleotide sequence described in any of 1 to 34) e, 1988, 239, 487.).
  • cDNA it is prepared by preparing mRNA from a plant by a conventional method (Maniatis et al. Molecular Cloning Cold Spring Harbor Laboratry Press), performing a reverse transcription reaction, and performing PCR using the same primers as described above. Is possible.
  • genomic DNA or cDNA a genomic DNA library or cDNA library is prepared by a conventional method.
  • the base sequence of the DNA of the present invention (for example, any one of SEQ ID NOs: 1-34) is prepared against this library. It can also be prepared by screening using a probe synthesized based on the base sequence described in (1).
  • the base sequence of the obtained DNA can be easily determined by using, for example, “Sequencer Model 373” (manufactured by ABI).
  • DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk is described below in the "root base of Eucalyptus genus Camaldrensis" and the center of the trunk.
  • DNA whose expression level is increased on the xylem side compared to the phloem side “ The expression level on the xylem side compared to the phloem side, ”
  • the expression level is higher on the xylem side compared to the phloem side, "in the central trunk of Eucalyptus genus Camaldrensis compared to the phloem side.
  • the expression level rises on the xylem side, and in the trunk trunk base, the DNA expression level is the same on the phloem side and the xylem side '', ⁇ the phloem in the stem of Eucalyptus genus Camaldrensis and Eucalyptus globules DNA whose expression level is higher on the xylem side than on the other side ”,“ The expression level rises on the xylem side compared to the phloem side, and the tree level on the eucalyptus globulus trunk, compared to the phloem side.
  • the timber of the trunk trunk is more of the fiber length (characteristic) than the timber of the trunk trunk. Is long It is known that thicker cell walls are thicker and fibril inclination is looser, and the wood fiber ratio and specific gravity are higher (mature material).
  • the xylem of the trunk of the trunk base Among the wood fiber formation, the wood fiber length is shorter, the diameter is thinner, the cell wall is thinner, the fibril inclination is steep, and the wood fiber rate It is known that the specific gravity is low (it is an immature material). Therefore, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side on the trunk base of a single pot” is expressed or increased in the immature wood formation site. It can also be expressed as “decreasing DNA”. In addition, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side in the central part of the eucalyptus trunk” is expressed in “ It can also be expressed as “DNA”.
  • the direction of the xylem of the Grobles trunk from the xylem of the Camaldrensis trunk is the fiber of the wood fiber formation (pulp quality) It is known that the length and diameter are thick and the cell wall is thick and the fibril inclination is loose and the wood fiber ratio and specific gravity are high. Also, the xylem of the Camaldrensis trunk is shorter than the globules trunk, but the diameter of the fiber is shorter, the cell wall is thinner, the fibril inclination is steeper, and the wood fiber ratio and specific gravity are lower. It has been known.
  • DNA that has an increased or decreased expression level on the xylem side compared to the phloem side, compared to the trunk of the Eucalyptus genus Camaldrensis ⁇
  • the thin cell walls are thin, the fibril inclination is steep, the wood fiber rate and specific gravity are low, and the expression level is increased or decreased at the wood fiber formation site with low quality pulp characteristics. '' It can also be expressed.
  • DNA with an increased or decreased expression level on the xylem side compared to the phloem side on the trunk of Eucalyptus globulus means that “wood fiber length is longer and diameter is thicker than the cell wall. It can be expressed as ⁇ DNA whose expression level is increased or decreased in the part that forms wood fiber with high quality pulp characteristics that thick fibril inclination is gentle, wood fiber rate and specific gravity are high '' .
  • the phloem side means the outside of the vascularization layer, which is a meristem, and preferably means the scab formation site on the phloem side.
  • the scab formation site is a site that is in the middle of or is capable of splitting into phloem cells, phloem tubules, parenchyma cells, and pelvic cells.
  • the xylem side means the inner side of the vascular bundle forming layer, preferably the xylem side wood fiber forming site.
  • the wood fiber formation site is a wood fiber cell, temporary conduit, conduit In other words, it is a part that is in the middle of separating into parenchymal cells or the like or has the ability to separate the cells.
  • the trunk base means a generally called breast height, for example, in the case of a 5- year-old plant of Eucalyptus, it indicates a ground height of less than about lm.
  • the central part of the trunk generally refers to the middle part of the culm height, for example, in the case of a eucalyptus fifth-year plant that has a culm height of 10 m or more, it refers to a height of about 5 m above the ground.
  • the DNA of the present invention or a partial DNA thereof can be used as a marker for examining the growth state of a trunk trunk (preferably a trunk of a trunk trunk or a middle section of a trunk) of a plant. That is, by using at least one of the above-mentioned DNAs or partial DNAs thereof, the growth state of the trunk trunk of the plant (preferably the trunk of the trunk trunk or the central part of the trunk) can be examined.
  • the trunk trunk portion of the plant is a xylem portion in which wood fiber formation is active. Furthermore, in the case of (2), it is determined that the trunk stem portion of the plant is a xylem portion in which immature wood is formed or a xylem in which immature material is formed in the future.
  • the immature wood means a wood fiber that is differentiated and formed from an immature formed layer structure, and the length of the wood fiber is shorter than that of the xylem of the center of the trunk collected under the same conditions.
  • the diameter is thin.
  • the cell wall is thin. The fibril inclination is steep, and the wood fiber ratio and specific gravity are low.
  • the trunk trunk of the plant is a xylem where mature wood is formed, or a xylem where mature wood is formed in the future.
  • matured wood means wood fibers that are formed by differentiation from a mature formation layer structure, and the length of the wood fibers is longer than that of the trunk of the trunk base collected under the same conditions.
  • the cell diameter is thick, the cell wall is thick, the fiber inclination is loose, and the wood fiber ratio and specific gravity are high and low.
  • the plant used in the test of the present invention is preferably Eucalyptus, more preferably Eucalyptus Camaldlensis or Eucalyptus Globulus.
  • the maturity qualitatively in the subject trunk Fibers are formed, or are formed in the future, and the amount of wood fibers ( It can be predicted that there will be a large (increase in specific gravity) or increase in the future (an average specific gravity of 450 to 500 kg / m 3 or more).
  • the material quality prediction as described above will clearly reveal the quality of the plant's growth, so if it is judged to be bad, fertilizer is applied in an oligotrophic area or weeding work is carried out if weeds are thick. It is possible to conduct operations quickly and appropriately, such as early implementation or thinning if the planting density is high.
  • material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance as shown in this application. .
  • the phloem in the trunk trunk and the middle trunk of the Eucalyptus genus Camaldrensis On the phloem side and the xylem side of the trunk of the plant (preferably the trunk trunk or the middle of the trunk), “the phloem in the trunk trunk and the middle trunk of the Eucalyptus genus Camaldrensis”
  • the expression level of at least one (preferably a plurality, more preferably all, the same below) of DNA whose expression level is increased on the xylem side relative to the xylem side is detected.
  • the expression level of the DNA on the trunk of the plant is higher on the xylem side than on the phloem side
  • DNA having an increased expression level on the xylem side compared to the phloem side in the trunk base and middle trunk of Eucalyptus genus Camaldrensis Of (a) to (e), or any of the DNAs described in any of them.
  • SEQ ID NO: 35-40, 42, 43, 45-48, 50, 51, 53-59, 61, 62, 64, 66 Is a DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of any one of 67
  • the expression level is reduced on the xylem side in the trunk base of Eucalyptus genus Camaldrensis, compared to the phloem side, and in the middle part of the trunk, the xylem side is compared with the phloem side.
  • Examples of the DNA whose expression level is increased in (1) include the DNAs described in any one of the following (a) to (e).
  • the expression level is increased on the xylem side compared to the phloem side in the central trunk of Eucalyptus genus Camaldrensis, and the expression level is increased on the phloem side and xylem side in the trunk base.
  • “equivalent DNA” include the DNAs described in the following (a) to (e)!
  • the base sequence ability D ⁇ described in any of SEQ ID NOs: 7, 10, 18, 26, 29, or 34 includes the bases described in any of SEQ ID NOs: 7, 10, 18, 26, or 29.
  • DNA comprising the sequence is preferred DNA comprising the base sequence described in SEQ ID NO: 18, 26 or 29 is more preferred! /, But is not limited to these! /.
  • the present invention provides a method for examining the growth state (maturity) of two different trunks. In other words, it provides a method for examining which of two different trunks are mature or immature.
  • “two different trunk trunks” include two trunk trunks in two different plants and two different trunk trunks in one plant. The above method can also be used to select mature or immature trunk trunks from a plurality of trunk trunks.
  • trunk trunk trunk trunk The other trunk tree section (hereinafter referred to as trunk trunk section B) is a mature section. Compared with trunk trunk section B, trunk trunk section A is an immature section. It is determined that there is. Therefore, the following (5) or (6) is an index for selecting an excellent line called an elite tree or a plus tree, for example, at a breeding site. At present, selection of elite trees, etc. is performed using the numerical values such as the diameter of the spiders and diameters and the material analysis values after harvesting as indicators, and there is no technique (concept) to select with the above indicators.
  • the “DNA having the nucleotide sequence described in SEQ ID NO: 33 or 34” is preferably a DNA consisting of the nucleotide sequence described in SEQ ID NO: 33, but is not limited to these! /.
  • (6) At least one DNA in two different trunk trunks, "DNA whose expression level is reduced on the xylem side of the trunk base compared to the xylem side of the eucalyptus trunk center" As a result of comparing the expression level of the DNA in two different trunk trunks, the expression level of the DNA decreased in the trunk trunk A compared to the trunk trunk B.
  • the DNA whose expression level is reduced on the xylem side of the trunk base compared to the xylem side of the eucalyptus trunk center is as follows: (a) to (e )! Or any of the DNAs listed elsewhere.
  • the trunk trunk portion of the plant is a xylem portion in which wood fiber formation is active. Furthermore, in the case of (8) or (9), the length of the fiber of the trunk of the plant is shorter and the diameter of the cell wall is narrower than that of the grops of the trunk of the groprus collected under the same conditions. The fibril inclination angle is steep, the wood fiber ratio and specific gravity are low, and V, a wood fiber having a pulp characteristic is formed! It is determined that the xylem is formed.
  • the length of the tree fiber is longer and the cell wall is thicker than the length of the stem of the stem of the plant, compared to the xylem of the Camaldrensis trunk collected under the same conditions. It is a xylem where wood fibers with excellent pulp properties are formed, such as the ratio of loose fibrils is high and the specific gravity is high, or the xylem where wood fibers with the pulp properties are formed in the future It is determined.
  • the material quality prediction as described above makes it clear that the state of plant growth is good or bad, so if it is judged to be bad, fertilization is performed in an oligotrophic area, or if weeds are overgrown, early weeding work is performed. If the planting density is high, thinning can be performed quickly and appropriately.
  • material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance.
  • DNA having an increased expression level on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules includes the following (a ) To (e)!
  • DNA having the nucleotide sequence described in any of SEQ ID NO: 11, 12, 17, 21, 22 or 30 includes the nucleotide sequence described in any of SEQ ID NO: 11, 12, 17, 22 or 30.
  • DNA consisting of the base sequence described in any one of SEQ ID NOs: 11, 12, 17 or 22 is more preferred, but is not limited thereto.
  • the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side.
  • DNA consisting of the base sequence described in any of SEQ ID NO: 8, 13, 19, 23-25 or 33 is the sequence described in any of SEQ ID NO: 8, 13, 19, 24, 25 or 33 DNA consisting of a base sequence is preferred, but DNA consisting of the base sequence described in any one of SEQ ID NOS: 13, 19, or 25 is more preferred, but is not limited thereto.
  • the expression level is increased on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis, and the expression level on the phloem side and xylem side in the trunk of the Eucalyptus globules
  • Examples of the DNA having the same DNA include DNAs described in any of the following (a) to (e).
  • DNA consisting of the base sequence described in the IJ number: 1-6, 9, 14, 16, 20, 27, 28 or 32! DNA sequence ability described in any of 6, 9, 14, 16, 20, or 27 is also preferred by DNA. SEQ ID NO: 1, 3, 6, 14, or 16 Although DNA is more preferable, it is not limited to these.
  • the present invention provides a method for inspecting the growth state (pulp quality) of the stem portion of two different plants (preferably eucalyptus). In other words, it provides a method for inspecting which of two different plant trunks is superior or inferior in pulp quality. This method can also be used to select good or inferior trunk trunks from multiple plant trunk trunks.
  • (11) or (12) it is compared with the trunk trunk of one of the two different plants (hereinafter referred to as plant A).
  • the stem of the other plant (hereinafter referred to as plant B) is a xylem with excellent pulp quality.
  • the xylem has Therefore, the following (11) or (12) is an index for selecting an excellent line called an elite tree or a plus tree at a breeding site, for example. At present, selection of elite trees, etc., is based on apparent values such as height and diameter, and material analysis values after cutting. There is no technology (concept) to select with the above indicators.
  • the eucalyptus is compared with the xylem side of the trunk of the Eucalyptus globulus.
  • the expression level of at least one of the DNAs whose expression level is increased on the xylem side of the trunk of the genus Camaldrensis is detected, and the expression level of the DNA in the trunk xylem of two different plants.
  • the expression level of the DNA is higher in the plant A than in the plant B.
  • the Eucalyptus genus Camaldrensis is compared with the xylem side of the trunk of the Eucalyptus globules.
  • Examples of “DNA having an increased expression level on the xylem side of the trunk” include the following DNAs (a) to (e)!
  • DNA consisting of the nucleotide sequence set forth in any of SEQ ID NO: 11, 12, 17 or 22 is preferably the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 12 or 17 Any of the DNAs having base sequence strengths described in any one of these is more preferable, but not limited thereto.
  • the eucalyptus genus Camaldrensis is compared with the xylem side of the trunk of the Eucalyptus globulus.
  • the expression level of at least one DNA of ⁇ DNA with decreased expression level at the xylem of the trunk of the tree '' was detected, and the expression level of the DNA in the trunk of the two different plants was compared.
  • the expression level of the DNA is reduced in the plant A compared with the plant B.
  • the tree of the Eucalyptus genus Camaldlensis is compared with the tree side of the Eucalyptus globulus. Examples of the “DNA whose expression level is reduced on the part side” include the DNAs described in (a) to (e) below, or any of them.
  • a xylem corresponding to any of the above (1) to (12) or a plant having the xylem is selected, and the selected xylem or the xylem It is possible to produce a uniform pulp from plants that have a large amount of pulp, making it possible to increase the efficiency of pulp production, reduce costs, and produce paper with new characteristics.
  • a xylem corresponding to any of the following (1) to (12) or a plant having the xylem according to a method well known to those skilled in the art, between the selected xylems or having the xylem Pulp can also be produced after confirming whether the wood fiber state is uniform among plants.
  • the present invention also provides a DNA encoding a plant transcription factor and a promoter DNA.
  • DNA encoding a plant transcription factor in the present invention can be used to control the formation of plant fiber fibers.
  • the promoter DNA of the DNA can be used to control expression specific to wood fiber tissue or wood fiber formation time.
  • the expression (level) information of DNA encoding a protein having a function to control the formation of plant fiber in the tree trunk is based on the quantitative state of the plant growth state, tree fiber formation state, and material quality. It can be used for measurement. Therefore, by using these, artificial modification of tree fiber cell shape formation, tree fiber cell-specific expression of any gene / protein, plant growth state, tree fiber formation state, quantitative quantity of materials It can be used for qualitative advance prediction. As a result, more efficient pulp production, reduced costs, and paper with new characteristics Can be manufactured.
  • Controlling wood fiber formation in plants has various important implications in the fields of industry and agriculture. For example, modification of the wood fiber properties of Eucalyptus genus Camaldrensis is significant in terms of improving the fiber properties of fiber raw materials such as pulp by increasing the fiber length. In addition, increasing the cellulose and hemicellulose content of Eucalyptus globulae species increases the yield of pulp and the like, and improves the cooking efficiency, which is significant in terms of economy and profitability.
  • the plant from which the DNA of the present invention is derived is not particularly limited, for example, useful crops (including forage crops) such as cereals, vegetables and fruit straw, fiber raw material plants such as pulp, ornamental plants, etc.
  • useful crops including forage crops
  • examples include plants.
  • the plant such as eucalyptus, pine, acacia, poplar, cedar, cypress, bamboo, yew, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower,
  • Examples include ivy, orange, grape, peach, pear, apple, tomato, Chinese cabbage, cabbage, Japanese radish, carrot, cabbage, cucumber, melon, parsley, orchid, chrysanthemum, lily and saffron.
  • examples of the DNA encoding a plant transcription factor include the DNAs described in any of the following (a) to (e).
  • stringent hybridization conditions include conditions of standing at 60 ° C in an O.lxSSC solution, or a stringency equivalent to this, An example is the condition. Under such conditions, DNA that hybridizes with DNA having the nucleotide sequence described in any one of SEQ ID NOs: 101 to 111 can be isolated.
  • DNA is extracted from a plant, a gene library is constructed, and screening is performed under the same conditions, or the extracted DNA is subjected to SEQ ID NO: 101-: L 11 From the base sequence described in any of the above, a contiguous neighboring sequence can be easily obtained by the TAIL-PCR method established by Liu et al. Using an arbitrary 20-mer sequence as a primer.
  • the present invention also provides a DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in any of SEQ ID NOs: 112 to 122.
  • Such DNA is preferably DNA encoding a protein having the structural characteristics described in the Examples.
  • a DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence of SEQ ID NO: 112 to 122 is generally isolated by a person skilled in the art by a known method. Is possible. For example, hybridization technology (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR) technology (Sai ki, RK. Et al, Science, 1985, 230, 1350. Saiki, RK. Et al, Science 1988, 239, 487.).
  • DNA having a base sequence ability described in any of SEQ ID NOs: 101 to L11 or a part thereof as a probe, or a DNA having a base sequence ability described in any of SEQ ID NOs: 101 to 111 It is known to those skilled in the art to isolate a DNA having high homology with a DNA having a nucleotide sequence described in any one of SEQ ID NOS: 101 to 111 from a plant using an oligonucleotide that specifically hybridizes as a primer. It is about normal deeds.
  • a hybridization reaction is preferably performed under stringent conditions.
  • stringent hybridization conditions refer to conditions of 6M urea, 0.4% SDS, 0.5 X SSC or equivalent stringency hybridization conditions. It is expected that DNA with higher homology can be isolated under conditions with higher stringency, for example, 6M urea, 0.4% SDS, and 0.1X SSC.
  • the DNA thus isolated has the SEQ ID NO: 101 at the amino acid level. It is considered to have a high homology with an amino acid sequence encoded by DNA having the nucleotide sequence ability described in any of ⁇ 111. High homology means at least 50% or more of the entire amino acid sequence, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, more preferably 95% or more, and most preferably 98% or more. Refers to sequence identity.
  • BLAST and Gapped BLAST programs the default parameters of each program are used. The technique is known (http://www.ncbi.nlm.nih.gov/).
  • the DNA of the present invention includes an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence of any one of SEQ ID NOs: 112 to 122.
  • Modification of amino acids in a protein is usually within 50 amino acids of all amino acids, preferably within 30 amino acids, more preferably within 10 amino acids, and even more preferably within 3 amino acids.
  • the amino acid modification can be performed using, for example, “Transformer Site—directed Mutagenesis Kit” or “ExSite PCR—Based 3 ⁇ 4ite—directed Mutagenesis KitJ (Clontech), for mutation or substitution, Deletion can be performed using “Quantum le ap Nested Deletion Kit J (Clontech)”.
  • the DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, cDNA, chemically synthesized DNA, and the like.
  • Genomic DNA is, for example, a genomic DNA prepared according to the method described in the literature (Rogers and Bendich, Plant Mol. Biol, 1985, 5, 69.) as a saddle type, and the base sequence of the DNA of the present invention (for example, sequence It can be prepared by performing PCR (Saiki et al.
  • a genomic DNA library or cDNA library is prepared by a conventional method.
  • the nucleotide sequence of the DNA of the present invention (for example, any one of SEQ ID NOs: 101 to 111) is prepared. It is also possible to prepare by screening using a probe synthesized based on the base sequence described above. The base sequence of the obtained DNA can be easily determined by using, for example, “Sequencer Model 373j (manufactured by ABI)”.
  • the DNA encoding the plant transcription factor is described below in the xylem side compared to the phloem side in the trunk base and middle trunk of Eucalyptus genus Camaldrensis.
  • the expression level rises in DNA ”,“ In the central trunk of Eucalyptus genus Camaldrensis, the expression level increases on the xylem side compared to the phloem side, and in the trunk base, The expression level on the xylem side is higher than that on the phloem side in the trunks of eucalyptus chamaldrensis and eucalyptus globulus.
  • DN A “ In the trunk of Eucalyptus genus Camaldrensis, the expression level increased on the xylem side compared to the phloem side, and in the trunk of the Eucalyptus globulus, the xylem side compared to the phloem side.
  • the xylem of the trunk of the trunk base Among the wood fiber formation, the wood fiber length is shorter, the diameter is thinner, the cell wall is thinner, the fibril inclination is steep, and the wood fiber rate It is known that the specific gravity is low (it is an immature material). Therefore, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side on the trunk base of a single pot” is expressed or increased in the immature wood formation site. It can also be expressed as “decreasing DNA”. In addition, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side in the central part of the eucalyptus trunk” is expressed in “ It can also be expressed as “DNA”.
  • the direction of the xylem of the Grobles trunk from the xylem of the Camaldrensis trunk is the fiber of the wood fiber formation (pulp quality) It is known that the length and diameter are thick and the cell wall is thick and the fibril inclination is loose and the wood fiber ratio and specific gravity are high. Also, the xylem of the Camaldrensis trunk is shorter than the globules trunk, but the diameter of the fiber is shorter, the cell wall is thinner, the fibril inclination is steeper, and the wood fiber ratio and specific gravity are lower. It has been known.
  • DNA that has an increased or decreased expression level on the xylem side compared to the phloem side, compared to the trunk of the Eucalyptus genus Camaldrensis ⁇
  • the thin cell walls are thin, the fibril inclination is steep, the wood fiber rate and specific gravity are low, and the expression level is increased or decreased at the wood fiber formation site with low quality pulp characteristics. '' It can also be expressed.
  • DNA with an increased or decreased expression level on the xylem side compared to the phloem side on the trunk of Eucalyptus globulus means that “wood fiber length is longer and diameter is thicker than the cell wall. It can be expressed as ⁇ DNA whose expression level is increased or decreased in the part that forms wood fiber with high quality pulp characteristics that thick fibril inclination is gentle, wood fiber rate and specific gravity are high '' .
  • the phloem side means the outside of the vascularization layer that is a meristem, and preferably the scab formation site on the phloem side.
  • the scab formation site is a site that is in the middle of or is capable of splitting into phloem cells, phloem tubules, parenchyma cells, and pelvic cells.
  • the xylem side means the inner side of the vascular bundle forming layer, preferably the xylem side wood fiber forming site.
  • the term “wood fiber forming site” refers to a site that is being divided into wood fiber cells, temporary conduits, conduits, parenchymal cells, etc., or that has the ability to separate them.
  • the trunk base means a generally called breast height, for example, in the case of a 5- year-old plant of Eucalyptus, it indicates a height of less than about lm above the ground.
  • the central part of the trunk generally refers to the middle part of the culm height, for example, in the case of a eucalyptus fifth-year plant that has a culm height of 10 m or more, it refers to a height of about 5 m above the ground.
  • the DNA of the present invention or a partial DNA thereof can be used as a marker for examining the growth state of a trunk of a plant (preferably, a xylem of a trunk trunk or a middle part of a trunk). That is, by using at least one of the above-mentioned DNAs or partial DNAs thereof, the growth state of the trunk trunk of the plant (preferably the trunk of the trunk trunk or the central part of the trunk) can be examined.
  • the trunk trunk portion of the plant is a xylem portion in which wood fiber formation is active. Further, in the case of (2), it is determined that the trunk trunk portion of the plant is a xylem portion where mature wood is formed, or a xylem where mature wood is formed in the future.
  • mature wood means wood fibers that are formed by differentiation from a mature formation layer structure, and the wood fiber length is longer than that of the trunk of the trunk base collected under the same conditions.
  • the cell wall is thick, the cell wall is thick, the fibril inclination is loose, and the wood fiber ratio and specific gravity are high.
  • immature wood means wood fibers that are differentiated and formed from an immature formed layer structure, and the length of the wood fiber is shorter than that of the wood part of the central trunk collected under the same conditions.
  • the cell wall is thin and the fibril inclination is steep, and the wood fiber rate and specific gravity are low.
  • the plant used in the test of the present invention is preferably Eucalyptus, more preferably Eucalyptus Camaldlensis or Eucalyptus Globulus.
  • the maturity qualitatively in the subject trunk Fibers are formed, or are formed in the future, and the amount of wood fibers ( It can be predicted that there will be a large (increase in specific gravity) or increase in the future (an average specific gravity of 450 to 500 kg / m 3 or more).
  • the material quality prediction as described above will clearly reveal the quality of the plant's growth, so if it is judged to be bad, fertilizer is applied in an oligotrophic area or weeding work is carried out if weeds are thick. It is possible to conduct operations quickly and appropriately, such as early implementation or thinning if the planting density is high.
  • material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance as shown in this application. .
  • the phloem in the trunk trunk and the middle trunk of the Eucalyptus genus Camaldrensis On the phloem side and the xylem side of the trunk of the plant (preferably the trunk trunk or the middle of the trunk), “the phloem in the trunk trunk and the middle trunk of the Eucalyptus genus Camaldrensis”
  • the expression level of at least one (preferably a plurality, more preferably all, the same below) of DNA whose expression level is increased on the xylem side relative to the xylem side is detected.
  • the expression level of the DNA on the trunk of the plant is higher on the xylem side than on the phloem side
  • DNA having an increased expression level on the xylem side compared to the phloem side in the trunk base and middle trunk of Eucalyptus genus Camaldrensis Of (a) to (e), or any of the DNAs described in any of them.
  • SEQ ID NO: 113-117 which encodes a protein having an amino acid sequence ability in which one or more amino acids are substituted, deleted, added, and Z or inserted in any one of the amino acid sequences described in any one of DNA
  • DNA having the base sequence ability described in any one of SEQ ID NOs: 102, 105 or 106 is preferable, and SEQ ID NO: 105 or DNA having a nucleotide sequence ability described in 106 is more preferable, but is not limited thereto.
  • the expression level is increased on the xylem side compared to the phloem side in the central trunk of Eucalyptus genus Camaldrensis, and the expression level is increased on the phloem side and xylem side in the trunk base.
  • “equivalent DNA” include the DNAs described in the following (a) to (e)!
  • DNA consisting of the nucleotide sequence of SEQ ID NO: 101, 107 or 109 to 111 DNA consisting of the nucleotide sequence of SEQ ID NO: 107 or 109 to 111 is preferred.
  • DNA having the nucleotide sequence set forth in SEQ ID NO: 107 or 109 is more preferred, but is not limited to these! /.
  • the present invention provides a method for examining the growth state (maturity) of two different trunks. In other words, it provides a method for examining which of two different trunks are mature or immature.
  • “two different trunk trunks” include two trunk trunks in two different plants and two different trunk trunks in one plant. The above method can also be used to select mature or immature trunk trunks from a plurality of trunk trunks.
  • trunk trunk A The xylem (hereinafter referred to as “trunk trunk B”) is a mature xylem, and compared to “trunk trunk timber B”, the trunk xylem A is determined to be an immature xylem.
  • Trunk trunk B is a mature xylem, and compared to “trunk trunk timber B”, the trunk xylem A is determined to be an immature xylem.
  • the following (3) is an index for selecting excellent lines called elite trees and brass trees, for example, in breeding sites. At present, selection of elite trees and the like is performed using an apparent numerical value such as the diameter of a spider and a material analysis value after cutting, and there is no technique (concept) for selecting with the above index.
  • DNA whose expression level is reduced on the xylem side of the trunk base as compared with the xylem side of the eucalyptus trunk center is the following (a) to (e): !, The DNA described in somewhere. (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 113 or 114
  • the “DNA having the nucleotide sequence ability described in SEQ ID NO: 102 or 103” is preferably a DNA having the nucleotide sequence ability described in SEQ ID NO: 102, but is not limited thereto.
  • the trunk stem portion of the plant is a xylem portion in which wood fiber formation is fostering.
  • the length of the fiber of the trunk of the plant is shorter and the diameter of the cell wall is narrower than that of the grops trunk of the groprus collected under the same conditions.
  • the fibril inclination angle is steep, the wood fiber ratio and specific gravity are low, and V, a wood fiber having a pulp characteristic is formed!
  • the length of the tree fiber is longer and the cell wall is thicker than the length of the stem of the trunk of the plant compared to the xylem of the Camaldrensis trunk collected under the same conditions. It is a xylem where wood fibers with excellent pulp properties are formed, such as the ratio of loose fibrils is high and the specific gravity is high, or the xylem where wood fibers with the pulp properties are formed in the future It is determined.
  • the material quality prediction as described above makes it clear that the state of plant growth is good or bad, so if it is judged to be bad, fertilization is performed in an oligotrophic area, or if weeds are overgrown, early weeding work is performed. If the planting density is high, thinning can be performed quickly and appropriately.
  • material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance.
  • DNA having an increased expression level on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules includes the following (a ) To (e)!
  • the “DNA having a nucleotide sequence ability described in SEQ ID NO: 103 or 105” is preferably a DNA having a nucleotide sequence ability described in SEQ ID NO: 105, but is not limited thereto.
  • the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side.
  • the expression level rises on the part side, and the expression level decreases on the xylem side compared to the phloem side in the trunk of Eucalyptus globules!
  • the DNA having the nucleotide sequence ability described in SEQ ID NO: 104 or 106 is preferably the DNA having the nucleotide sequence ability described in SEQ ID NO: 106, but is not limited thereto.
  • the expression level is increased on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis, and the expression level on the phloem side and xylem side in the trunk of the Eucalyptus globules
  • Examples of the DNA having the same DNA include DNAs described in any of the following (a) to (e).
  • the tree of the Eucalyptus genus Camaldrensis is compared to the phloem side.
  • the expression level of at least one DNA is detected in the eucalyptus globulus trunk, the expression level of which is reduced on the xylem side compared to the phloem side.
  • the growth state of the trunk stem part of two different plants is compared with the phloem side.
  • a method for inspecting pulp quality is provided. In other words, it provides a method for inspecting which of two different plant trunks is superior or inferior in pulp quality. This method can also be used to select good or inferior trunk trunks from multiple plant trunk trunks.
  • (11) it is compared with the trunk trunk of one of the two different plants (hereinafter referred to as plant A).
  • the trunk of the other plant (hereinafter referred to as plant B) is a xylem having an excellent pulp quality.
  • the trunk xylem A has an inferior pulp quality. It is determined that the xylem has. Therefore, the following (11) is an index when selecting excellent lines called elite trees and plus trees, for example, at breeding sites. At present, selection of elite trees, etc. is performed using the numerical values such as the diameter and the material analysis values after cutting, and there is no technology (concept) to select using the above indicators.
  • the eucalyptus is compared with the xylem side of the trunk of the Eucalyptus globulus.
  • the expression level of genus Camaldrensis is reduced at the xylem side of the trunk, and the expression level of at least one of the DNA is detected, and the expression level of the DNA in the trunk xylem of two different plants is detected.
  • the expression level of the DNA is reduced in plant A compared to plant B.
  • the Eucalyptus genus Camaldrensis is compared with the xylem side of the trunk of Eucalyptus globules.
  • Examples of the “DNA whose expression level is reduced on the xylem side of the trunk” include the following (a) to (e)!
  • a xylem corresponding to any of the above (1) to (8) or a plant having the xylem is selected, and the selected xylem or the xylem is selected. It is possible to produce a uniform norp from plants with xylem, making it possible to increase the efficiency of pulp production, reduce costs, and manufacture paper with new characteristics.
  • Pulp can also be produced after confirming whether the wood fiber state is uniform among plants having the. Further, if it does not fall under any of the following (1) to (8), it is obtained by collecting a xylem or a plant having the xylem to produce a pulp having an arbitrary composition.
  • the test of the present invention can be carried out by various known methods. For example, first, an RNA sample is prepared from a desired tissue of a test plant. Next, the amount of target RNA contained in the RNA sample is measured. The amount of RNA measured is then compared to a control. Examples of such methods include Northern blotting and RT-PCR.
  • the test of the present invention can also be performed by a DNA array method.
  • examples of the nucleotide immobilization (array) method include an oligonucleotide-based array developed by Aflfymetrix.
  • oligonucleotide array oligonucleotides are usually synthesized in situ.
  • photolithographic technology Alfymetrix
  • inkjet Rosetta Inpharmatics
  • the “substrate” means a plate-like material on which a nucleotide probe can be fixed.
  • the substrate of the present invention is not particularly limited as long as the nucleotide probe can be immobilized, but a substrate generally used in DNA array technology is preferably used. it can.
  • a DNA array consists of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate.
  • a force permeable membrane which is generally glass, can be used, for example, a trocellulose membrane.
  • the nucleotide probe immobilized on the substrate is not particularly limited as long as it can detect the expression of the DNA of the present invention. That is, the probe is a probe that hybridizes to the DNA of the present invention. If specific hybridization is possible, the nucleotide probe need not be perfectly complementary to the DNA of the present invention.
  • the length of the nucleotide probe to be bound to the substrate is not particularly limited, but is usually 10 to 100 bp, preferably 10 to 50 bp, more preferably 15 to 25 bp.
  • the DNA of the present invention is brought into contact with the substrate. Through this process, the DNA of the present invention is hybridized to the nucleotide probe.
  • the hybridization reaction solution and reaction conditions may vary depending on various factors such as the length of the nucleotide probe immobilized on the substrate, it can be generally performed by methods well known to those skilled in the art.
  • the intensity of hybridization between the DNA of the present invention and the nucleotide probe immobilized on the substrate is then detected.
  • This detection can be performed, for example, by reading the fluorescence signal of the fluorescent dye labeled on the DNA of the present invention with a scanner or the like.
  • the present invention also provides a promoter DNA of the DNA of the present invention.
  • the promoter DNA of the present invention includes a promoter DNA linked to a gene obtained by the present invention, in particular, a gene having a differential expression in the spatial position of an oak.
  • promoter-one DNA means DNA containing a specific base sequence necessary for the initiation of synthesis (transcription) of mRNA using DNA as a saddle type. It includes DNA created by artificial alterations such as replacement.
  • the promoter DNA of the present invention includes a protein having a function of forming a tree fiber cell wall of a plant trunk (a protein involved in cellulose synthesis and a protein involved in ligne synthesis).
  • Examples include promoter DNA of encoding DNA and promoter DNA of DNA encoding plant transcription factor.
  • Plant trunk tree fiber cells The promoter DNA of DNA encoding a protein having a function of forming a wall (a protein involved in cellulose synthesis and a protein involved in lignin synthesis) is shown in SEQ ID NO: 93-: LOO. Proteins that have the function of forming tree fiber cell walls of plant trunks (proteins involved in cellulose synthesis and proteins involved in lignin synthesis)
  • DNA encoding and its promoter DNA is as described in the Examples. Further, promoter DNAs of DNAs encoding plant transcription factors are shown in SEQ ID NOs: 156 to 166. The relationship between DNA encoding a plant transcription factor and its promoter DNA is also as described in the Examples.
  • the promoter of the present invention can be produced and used as follows, for example. Extract and purify DNA from the tissues of the desired eucalyptus plant. In preparing DNA, various methods can be used, and commercially available kits such as ISOPLANT kit (manufactured by Futtsubon Gene Co., Ltd.) can be used.
  • oligonucleotides were prepared from any two locations based on the base sequence of eucalyptus cDNA that had been successfully isolated by the present inventors and used as primers. By PCR, genomic DNA corresponding to the selected eucalyptus cDNA can be easily produced.
  • Upstream DNA of the gene is obtained by PCR using oligonucleotide primers created based on the base sequence of the gene (Inverse-PCR method or anchor PCR method ⁇ AIL-PCR method (supervised by Isao Shimamoto, PCR Experiment Protocol ”(plant cell engineering separate volume, plant cell engineering series 7) published by Shujunsha, July 1997), or by the hybridization method using the DNA sequence of the gene as a probe. Is possible.
  • a genomic DNA library can also be used for eucalyptus DNA.
  • various genome vectors TAC vectors (Liu et al. (1999), Proc. Natl. Acad. Sci. USA, vol96: p653 5) It is obtained by transforming E. coli.
  • Hybridization technology can be used for screening genomic DNA libraries.
  • the eucalyptus cDNA sequence that has been successfully isolated by the present inventors can be used.
  • the above genomic DNA library is screened to isolate a clone containing a DNA sequence homologous to the gene. Restriction Enzyme cleavage maps are created and nucleotide sequences are determined, the structure of the cloned DNA is clarified, and the sequence existing upstream of the gene is identified.
  • This upstream sequence preferably includes a TATA box sequence and is at least several hundred bp force of several kbp. This sequence is excised with an appropriate restriction enzyme and subcloned into another plasmid vector or the like as necessary.
  • the promoter activity of the above sequence can be analyzed as follows. For example, using a vector containing a reporter gene such as ⁇ , subcloning is performed so that the above sequence is linked upstream of the reporter gene.
  • the ⁇ vector uses E. coli / 3 dalc mouth-dase (GUS) as a reporter gene.
  • GUS E. coli / 3 dalc mouth-dase
  • this gene product uses 5-bromo-4-chloro-3-j8-D-glucronic acid (X-glucuric acid) as a substrate, it decomposes to produce indigotin, a blue precipitate. It is possible to monitor the current situation at the organizational level.
  • 4-methyHimbellif eryj8-D-glucronide When 4-methyHimbellif eryj8-D-glucronide (4MUG) is used as a substrate, gene expression can be quantified by fluorescence generated by the action of the gene product.
  • a chloramphee-cholacetyl transferase gene, a luciferase gene, a green fluorescein protein gene, and the like can be used as a reporter gene.
  • the chimeric gene construct prepared as described above can be introduced into plants such as Arabidopsis thaliana via agrobacterium and analyzed for its function.
  • pBI101 is used as a vector
  • a recombinant plasmid containing the chimeric gene is introduced into the MP90 strain of Agrobacterium tumefaciens, for example, using the electoporation method, and the resulting transformant
  • Arabidopsis thaliana plants are infected by the floral dip method (supervised by Isao Shimamoto et al., "Experimental protocol for model plants" (plant cell engineering separate volume, plant cell engineering series 4) Shujunsha published in April 1996).
  • Seeds obtained from the infected plant are sown in a medium containing a drug such as kanamycin based on the vector used to obtain a transformant that has become drug-resistant by gene transfer. Using this transformed substance, the expression of the reporter GUS gene is analyzed.
  • the promoter of the present invention or an expression vector containing the promoter can be used as follows.
  • An expression vector is constructed by inserting a chimeric gene in which genes involved in physical biosynthesis are linked, for example, into a ⁇ vector. This vector is introduced into, for example, Tabacco plants via agrobacterium.
  • the gene of the present invention is expected to be expressed at the site of tree fiber formation and to produce any secondary metabolite by the action of the promoter of the present invention. In this case, since there is no phenomenon that is expressed even in an unnecessary tissue like the 35S promoter, it is expected that other preferable traits do not appear.
  • the gene that can be controlled by the promoter of the present invention is not limited to the specific gene described above. It is also possible to modify the function of the promoter of the present invention by linking other expression control sequences to the promoter of the present invention. Examples of such expression control sequences include enhancer sequences, repressor sequences, and insulator sequences.
  • the promoter of the present invention includes several cis-element sequences that control the expression of genes involved in the above-ground specific location of trunk and cell wall biosynthesis as functional characteristics. For the purpose of using the cis element sequence contained in the promoter of the present invention, it is possible to insert a part of the promoter of the present invention into another promoter and modify the function of the promoter.
  • the present invention also provides DNA for suppressing the expression of a DNA encoding a protein involved in plant fiber fiber wall formation.
  • DNA for suppressing the expression of an endogenous gene is preferred in that the DNA encoding an antisense RNA complementary to the transcription product of the DNA of the present invention and the transcription product of the DNA of the present invention are specifically used.
  • RNA encoding RNA with ribozyme activity to cleave DNA encoding RNA that suppresses expression of DNA of the present invention by RNAi effect or co-suppression effect, dominant negative for transcript of DNA of the present invention
  • Examples thereof include DNA encoding a protein having various traits.
  • the above “suppression of endogenous gene expression” includes suppression of gene transcription and suppression of translation into Z or a protein that also encodes the gene force. Also included is a decrease in expression as well as complete cessation of expression of the gene.
  • antisense nucleic acids suppress the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kogaku 2 Nucleic acid IV gene replication and expression, Japan biochemicalization) The Society, Tokyo Chemistry, 1993, 319-347.)
  • the antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above.
  • an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of a gene is designed, it is considered effective for inhibiting the translation of the gene.
  • a sequence complementary to the coding region or the 3 ′ untranslated region can also be used.
  • DNA containing an antisense sequence of not only a translation region of a gene but also an untranslated region is also included in the antisense DNA used in the present invention.
  • the antisense DNA to be used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.
  • the DNA prepared in this way can be By using the method, it can be transformed into a desired plant.
  • the sequence of the antisense DNA is preferably a sequence complementary to an endogenous gene or a part of the plant to be transformed, but is completely complementary as long as the gene expression can be effectively suppressed. It doesn't have to be.
  • the transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene.
  • the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. .
  • the length of the antisense DNA usually used is shorter than 5 kb, preferably shorter than 2.5 kb.
  • Ribozyme refers to an RNA molecule that has catalytic activity.
  • research focused on ribozymes as enzymes that cleave RNA has enabled the design of ribozymes that cleave RNA in a site-specific manner.
  • Some ribozymes have a size of 400 nucleotides or more, such as the group I intron type and Ml RNA contained in RNase P, but the hammerhead type has an active domain of about 40 nucleotides called the hairpin type.
  • the self-cleaving domain of the hammerhead ribozyme has the ability to cleave the 3 'side of C15 in the sequence G13U14C15.
  • base pairing between U14 and A9 is important. It has been shown that A15 or U15 can also be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.) 0 Design a ribozyme whose substrate binding site is complementary to the RNA sequence near the target site
  • a restriction enzyme-like RNA cleavage ribozyme that recognizes the sequence UC, UU, or UA in the target RNA can be generated (Koizumi, M.
  • Hairpin ribozymes are also useful for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that target-specific RNA cleavage ribozymes can also be generated from hairpin ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.).
  • the ribozyme designed to cleave the target is linked to a promoter and transcription termination sequence, such as the cauliflower mosaic virus 35S promoter, so that it is transcribed in plant cells. At this time, if an extra sequence is added to the 5 'or 3' end of the transcribed RNA, the activity of the ribozyme may be lost. In this case, the RNA containing the transcribed ribozyme It is possible to place another trimming ribozyme that acts on cis on the 5 'side or 3' side of the ribozyme part (T aira, K. et al, Protein Eng, 1990, 3, 733., Dzianott, AM.
  • RNAi RNA interference
  • RNAi refers to a phenomenon in which the expression of the introduced foreign gene and target endogenous gene and the deviation are suppressed when a double-stranded RNA having the same or similar sequence as the target gene sequence is introduced into the cell. .
  • the details of the RNAi mechanism are not clear, but it is thought that the target gene is degraded from the fact that the double-stranded RNA introduced first is broken down into small pieces and somehow serves as an indicator of the target gene. .
  • RNAi is known to be effective in plants (Chuang, CF.
  • RNAi RNA having a nucleotide sequence set forth in any of SEQ ID NOs: 1-34 or a sequence similar thereto. What is necessary is just to introduce
  • the gene used for RNAi is completely the same as the target gene.
  • the sequence identity is at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more. Further, the identity of the sequence can be determined by the method described above.
  • Suppression of endogenous gene expression can also be achieved by co-suppression caused by transformation of DNA having the same or similar sequence as the target gene sequence.
  • “Co-suppression” is a phenomenon in which when a gene having the same or similar sequence as a target endogenous gene is introduced into a plant by transformation, the expression of the introduced foreign gene and the target endogenous gene are both suppressed. Refers to that. The details of the mechanism of co-suppression are not clear, but at least part of the mechanism is thought to overlap with the RNAi mechanism. Co-suppression is also observed in plants! (Smyth, DR., Curr Biol, 1997, 7, R793., Martienssen, R., Curr Biol, 199 6, 6, 810.).
  • a vector prepared so that the DNA or a DNA having a similar sequence can be expressed.
  • the gene used for co-suppression need not be completely identical to the target gene, but at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more of sequence identity. Have.
  • the sequence identity can be determined by the method described above.
  • the suppression of the expression of the endogenous gene in the present invention can also be achieved by transforming a gene encoding a protein having a dominant negative trait to the protein encoded by the target gene into a plant.
  • a gene encoding a protein having a dominant negative trait refers to a gene having a function of eliminating or reducing the activity of an endogenous wild-type protein inherent in a plant by expressing the gene. Refers to that.
  • the present invention also provides a recombinant vector containing the DNA.
  • the vector of the present invention is not particularly limited as long as it contains a promoter sequence that can be transcribed in plant cells and a terminator sequence that includes a polyadenylation site necessary for the stability of the transcript.
  • pUC Vectors that can be amplified in E. coli, such as derivatives, and shuttle vectors that can be amplified in both E. coli and agrobacterium, such as ⁇ (Clontech). I can get lost.
  • plant viruses such as cauliflower mosaic virus can be used as vectors for ⁇ IJ.
  • the vector of the present invention can be obtained, for example, by binding or inserting the promoter DNA of the present invention or a promoter DNA for constitutively or inducibly expressing a desired gene into a predetermined part of the vector. it can.
  • the method for inserting the promoter into the vector follows the method for inserting a normal gene into the vector.
  • An expression vector for gene expression can be obtained by functionally connecting a desired gene to the promoter of this recombinant vector.
  • promoters for constitutive expression include the cauliflower mosaic virus 35S promoter (Odell et al., Nature, 1985, 313, 810.) and the rice actin promoter (Zhang et al., Plant). Cell, 1991, 3, 1155.) and corn ubiquitin promoter (Cornejo et al., Plant Mol. Biol, 1993, 23, 567.).
  • promoters for inducible expression are expressed by external factors such as invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, UV irradiation, and spraying of specific compounds. There are known promoters.
  • Such promoters include, for example, the rice chitinase gene promoter (Xu et al., Plant Mol. Biol., 1996, 30, 387.) and tapa PR protein gene promoters (Ohshima et al., Plant Cell, 1990, 2, 95.), rice-derived “lipl9” gene promoter (Aguan et al., Mol.
  • Corn alcohol dehydrogenase promoter (Walker et al "Proc. Natl. Acad. Sci. USA, 1987, 84, 6624.)
  • rice chitinase gene promoter and tobacco PR protein gene The promoter of certain compounds such as salicylic acid, and “ ra bl6” is a plant hormone It is also induced by application of abscisic acid.
  • the above recombinant vector contains an appropriate selection marker gene, or a plasmid vector containing a selection marker gene and the cell. It is preferable to introduce.
  • Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene that is resistant to the antibiotic hygromycin, the neomycin phosphotransferase that is resistant to kanamycin or gentamicin, and the gene that is resistant to the herbicide phosphinothricin. Examples include cetyltransferase gene.
  • the present invention also provides a transformed plant cell into which the vector of the present invention has been introduced.
  • the cells into which the vector of the present invention is introduced are not particularly limited, for example, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower, ivy, orange, grape , Peach, pear, apple, tomato, cabbage, cabbage, radish, carrot, cabotya, cucumber, melon, parsley, orchid, chrysanthemum, lily, safran, etc. Trees such as poplar, cedar, cypress, bamboo, and yew are desirable.
  • the plant cells of the present invention include cells in plant bodies. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots.
  • a target gene can be introduced into a plant by using a plant virus as a vector.
  • plant viruses that can be used include cauliflower mosaic virus. That is, first, after preparing the recombinant by inserting the viral genome into a vector derived from E. coli, these target genes are inserted into the viral genome. insert. It is possible to introduce these target genes into a plant by excising the viral genome thus modified from the recombinant with a restriction enzyme and inoculating the plant (Hohn et al. (1982) Molecular Biology of Plant Tumors (Academic Press ⁇ New York) pp549, US Pat. No. 4,407,956). Vectors introduced into plant cells and plants are not limited to these, and include other possibilities.
  • plasmids for direct introduction into protoplasts, there are no restrictions on the required vectors.
  • a simple plasmid such as a pUC derivative can be used.
  • other DNA sequences may be required.
  • Ri plasmids when ⁇ or Ri plasmids are used for transformation of plant cells, the sequence of at least the right end of the T-DNA region of Ti and Ri plasmids, usually the sequences at both ends, must be inserted into the gene to be introduced. Must be connected to be adjacent to the.
  • Agrobacterium When Agrobacterium is used for transformation, the gene to be introduced needs to be cloned into a special plasmid, that is, an intermediate vector or a binary vector. Intermediate vectors are not replicated in Agrobacterium.
  • the intermediate vector is transferred into the genus Agrobataterium by a helper plasmid or electoral position.
  • the intermediate vector has a region that is homologous to the T-DNA sequence, and is incorporated into the Agrobacterium sputum or Ri plasmid by homologous recombination.
  • Agrobacterium used as a host must contain the vir region. Usually, vir or Ri plasmid contains vir region, and T-DNA can be transferred to plant cells by its function.
  • a binary vector can be replicated and maintained in an Agrobacterium, so when incorporated into an Agrobacterium by the helper plasmid or the electroporation method, the vir region of the host By function, T-DN A on the binary vector can be transferred to plant cells.
  • the present invention also provides a transformed plant regenerated from the transformed plant cell, a transformed plant that is a descendant or clone of the transformed plant, and the transformed plant.
  • Such a transformed plant is a useful transformed plant in which cell wall components and cell morphogenesis are modified by plant species.
  • the modification of the cell wall component in the present invention is not particularly limited, for example, high cellulose, low lignin, a thick cell wall, a thin cell, a long fiber length, a short one, various quantitative and qualitative. Change. Examples of the modification of the cell morphology include, but are not limited to, changes in cell elongation and changes in cell size (quantitative change in volume).
  • the transformed plant according to the present invention has, for example, a plant having a new value such as an increase in the amount of plant growth due to an increase in the amount of cell wall synthesis, a change in fiber cell morphology, an increase in useful components of crops depending on the plant species.
  • a plant having a new value such as an increase in the amount of plant growth due to an increase in the amount of cell wall synthesis, a change in fiber cell morphology, an increase in useful components of crops depending on the plant species.
  • Useful as It is also useful as a plant with new value, such as the development of new materials by controlling cell wall synthesis, increased digestion and absorption efficiency of forage crops, and changes in fiber cell morphology.
  • the "transformed sickle object” is a plant having the above-described transformed sickle cell, and includes, for example, a transformed plant regenerated from the transformed cell. Be turned.
  • the method of regenerating an individual from transformed plant cells varies depending on the type of plant cell. For example, the method of Fujimura et al. (Fujimura et al, Plant Tissue Culture Lett., 2, 74, 1995) is used for rice, and Shillito is used for maize. (Shillito et al., Bio / Technology, 7, 581, 1989), in potato, in Visser et al., Theor. Appl.
  • the present invention introduces into a host cell an expression vector having a gene group involved in plant fiber cell wall formation in plants, particularly trees, or homologues thereof, or a promoter region linked to these genes.
  • the method includes the steps of obtaining a transformed cell, regenerating a transformed plant from the transformed cell, obtaining a plant seed from the obtained transformed plant, and producing the plant from the plant seed.
  • the process of obtaining plant seeds from the transformation and the object is, for example, the cultivation of the transformation and the object. It refers to the process of collecting from the ground, transplanting it to a pot containing water-containing soil, growing it at a constant temperature, forming flowers, and finally forming seeds.
  • the process of producing a plant from a seed is, for example, when the seed formed on a transformed plant matures, is isolated and sown in soil containing water, and grows under a constant temperature and illuminance. This refers to the process of producing a plant body.
  • the presence of the introduced foreign DNA or nucleic acid in the transformed plant body is determined by a known PCR method or Southern hybridization method, or by analyzing the nucleotide sequence of the nucleic acid in the plant body. Can be confirmed.
  • transformation and extraction of DNA or nucleic acid from the object can be performed according to the known method of J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor laboratory Press, 1989).
  • an amplification reaction is carried out using the nucleic acid extracted from the regenerated plant body as described above in a cage shape.
  • an amplification reaction can also be carried out in a reaction mixture in which a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of DNA of the present invention is used as a primer and these are mixed.
  • amplification reaction when DNA denaturation, annealing, and extension reactions are repeated several tens of times, an amplification product of a DNA fragment containing the DNA sequence of the present invention can be obtained.
  • the reaction solution containing the amplified product is subjected to, for example, agarose electrophoresis, it is possible to confirm that the amplified DNA fragments are fractionated and that the DNA fragments correspond to the DNA of the present invention. .
  • the present invention also provides a primer that amplifies all or a part of the base sequences set forth in SEQ ID NOs: 1-34 and 101-111.
  • the primer of the present invention can be used in the inspection method of the present invention.
  • the primer of the present invention is not particularly limited as long as it can amplify at least a part of the DNA of the present invention or its complementary strand. Is usually 15 bp to 100 bp, preferably 16 bp to 31 bp, and more preferably, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 31 nucleotides.
  • the 3 ′ region may be complementary, and a restriction enzyme recognition sequence or a tag may be added to the 5 ′ side.
  • the present invention provides a primer set for amplifying all or part of the base sequence described in SEQ ID NOs: 1-34, 101-: L11.
  • the primer set of the present invention includes:
  • PCR primer of the present invention can be prepared by those skilled in the art using, for example, an automatic oligonucleotide synthesizer.
  • the present invention also provides a reagent comprising the above primer set.
  • the reagent of the present invention can be used in the inspection method of the present invention.
  • Such reagents may include those used in the above-described detection methods.
  • the gene, primer, staining solution and the like of the present invention can be mentioned.
  • distilled water, salt, buffer solution, protein stabilizer, preservative and the like may be contained.
  • glucose is obtained from the lignocellulose obtained therefrom by an acid hydrolysis or enzymatic decomposition (cellulase) process, and then ethanol is obtained by alcohol fermentation.
  • acid hydrolysis or enzymatic decomposition cellulase
  • ethanol is obtained by alcohol fermentation.
  • biodegradable plastics polylactic acid
  • biomass derived from wood is expected to become the mainstream instead of cereal as food.
  • Lignin is also expected to be used for plastics and adhesives, although it will be necessary to overcome technical challenges in the future.
  • lignin is decomposed ichiologically in the pulp manufacturing process of the paper industry and is contained in the waste liquid (called black liquor). After extracting the necessary chemicals from the waste liquid, It is used as fuel in the factory. In other words, it is nothing else but to rely on woody biomass for a part of the fuel.
  • woody biomass is cultivated stably and on a large scale and circulated by afforestation, so that it can be used as a conventional raw material, as well as petroleum alternative energy by biomass conversion, and also cellulose or hemicellulose It is quite possible to create a new plastic from this (which is also technically possible). Furthermore, the spread of woody biomass will become a solution to energy security and environmental problems, and at the same time will lead to the development of new industries such as agriculture and forestry and the creation of employment opportunities.
  • eucalyptus tissue to be extracted For example, stem growth (secondary wall thickening zone; tissue rich in formation layer), leaves, roots, slant stimulation, stress application by salt solution exposure, etc. The gene expression from the situation is assumed.
  • the basic method of extraction is the method described by Hino et al. (Japanese Patent Application No. 6-219187).
  • Hino et al. Japanese Patent Application No. 6-219187.
  • an RNA extraction method using eucalyptus roots by hydroponics as a material will be described in detail.
  • 6Mo 2 O prepared with demineralized water, pH is 0.1M NaOH or KOH daily 6. Adjusted to 0. Furthermore, all the culture medium was changed every week. When stress treatment is performed, the culture solution supplemented with NaCl so that the final concentration is 50,100,200,300 mM in order from the first day to the fourth day of cultivation is controlled in the stress treatment area, and the culture solution without addition of NaCl is controlled. And On the fourth day, 10 g of the roots were cut into small pieces and ground in liquid nitrogen. This was transferred to a 50 ml centrifuge tube (manufactured by NUNC), 10 g of glass beads were added, and then ground for 5 minutes with a homogenizer.
  • NUNC 50 ml centrifuge tube
  • a methanol solution containing dithiothreitol (lmg / ml) was used for this, and solvent extraction of the ground sample was repeated until no coloration was observed in the supernatant (about 3 times). After extraction, the sample was lyophilized. This lyophilized sample was mixed with 25 ml of pH 9 lOOmM CHES buffer (20 milligrams of dithiothreitol and 10 mM vanadyl ribonucleoside compound solution added immediately before use) and incubated at 65 ° C for 30 minutes. A 5M sodium chloride solution and a 10% CTAB solution were added to the sample solution after the incubation.
  • the sodium chloride concentration in the sample solution after addition was 1.4 M, and the CTAB concentration was 1% (w / v).
  • RNA precipitate was collected by centrifugation.
  • 12M lithium chloride solution was added to a final concentration of 3M, mixed well, and then ice-cooled for 1 hour.
  • the RNA precipitate was collected by centrifugation, washed and dried, and finally dissolved in 100 L of water to obtain a total RNA fraction.
  • 610 g of total RNA was obtained from each of the roots of the stress-treated group and the control root.
  • PolyATtract mRNA Isolation Systemlll & IV kit Cat. # Z5300 & Z5310 Promega Corp. USA
  • 1.3 ⁇ g of the mRNA was obtained from 610 g of total RNA and 1.8 ⁇ g of mRNA was obtained from the control sample.
  • the eucalyptus mRNA derived from various tissues and situations obtained by the method shown in (1) was synthesized using the Clontech Smart cDNA library construction kit, and finally the eucalyptus mRNA was synthesized. -Dimid library.
  • Each cDNA library created in this way also had independent clone power of more than 10 X 10 6 pfo.
  • Library amplification was performed on a part of the library prepared, and the clone was analyzed without amplification.
  • genes related to cellulose and lignin synthesis (Proc Natl Acad Sci USA 2001 Dec 4; 98 (25): 14732-7., JP 2000-41685, JP 2002-95482, JP We searched for clones with high homology. As a result, 59 clones were identified. However, some of these clones include those involved in the formation of scab tissue and the like that are formed only by wood fiber formation and those that are not related to cell wall synthesis.
  • a eucalyptus oligo microarray was prepared from the eucalyptus EST database. The actual production was outsourced to Agilent Technologies, Inc. (Yokogawa Analytical Systems is the Japanese distributor). Details are described on the following website: o http: // www. Chem. Agilent ⁇ com / cag / country / JP / products / PL, ol494.htm
  • the eucalyptus oligo microarray produced in this way contains 8400 oligo DNAs, and can cover most of the genes that are expressed in eucalyptus trunks.
  • wood fibers Most of the woody biomass in the trunk is composed of tissue cells called wood fibers.
  • the main factors that determine the properties of wood fibers include the specific gravity of the wood fiber structure, the cell wall structure and components (cellulose, hemicellulose, lignin). These are formed by working together in a group of many genes that are not defined by a single gene or protein.
  • the properties of wood fibers are known to vary depending on the species of hardwood 'coniferous trees, high above ground'.
  • RNA extraction method shown in Example 1 the present inventors extracted total RNA from the trunk base and middle part of a 5-year-old Camaldrensis species, and Groplus species. At this time, the crust side (phloem) and the wood fiber-forming tissue side (xylem) of each plant were used. The total RNA power obtained from each individual was purified by the PolyATract mRNA Isolation System manufactured by Promega. Each cRNA obtained by synthesizing a total of 4 types of mRNA obtained in this way was labeled with 2 types of fluorescent dyes (cy3.cy5), and used as probes in oligo microarray analysis. An analysis was performed ( Figures 1-3).
  • the Sequence ID corresponds to SEQ ID NOs: 1-34 in order from the top.
  • CesA Cellulose synthase
  • SuSy Sucrose Synthase
  • EGase endo— 1,4— beta— glucanase
  • DL Dynamin-like protein
  • CAD cinnamyl alcohol dehydrogenase
  • OMT ⁇ — methyl transferase
  • 4CL 4— coumarate Co
  • CCR Cinnamoyl Co
  • reductase C4H
  • the microarray experiment 5y-cam5 X vs P is an experiment comparing the xylem and phloem of the middle part of the fifth-year eucalyptus / camaldolensis trunk, and 5y-caml X vs P
  • Experiment comparing phloem, 5y-glo X vs P, 5th grade Eucalyptus' Grobulus An experiment comparing the xylem and phloem of the base.
  • the experimental results are expressed as a positive multiple when the former is strong, and as a negative multiple when strong on the husk side, comparing the wood fiber-forming tissue side and the husk side.
  • the 34 gene groups were broadly classified into the following (1) to (3) based on the detailed expression patterns in the trunk base and the central part of the fifth-grade Camaldrensis species (Table 2, Four).
  • the 26 gene groups include gene groups (SEQ ID NOs: 1 to 6, 9) that have an expression differential power of 3 ⁇ 4 times or more on the xylem side compared to the phloem side at either the trunk base or the trunk trunk center. , 11-14, 1 6, 17, 19, 20, 22, 25, 27, 30), the trunk of the trunk or the center of the trunk of the tree! Gene group (SEQ ID NOs: 1, 3 to 6, 9, 11 to 14, 16, 17, 19, 22, 25, 30), stem base or trunk A group of genes (SEQ ID NOs: 1, 3-6, 11, 12, 14, 16, 17, 22, which are more than double the differential force on the xylem side compared to the phloem side in the! 25), a group of genes (SEQ ID NO: 1 ⁇ 3, 5, 12, 25) Power included.
  • the numerical value of the average fluorescence intensity obtained by the experiment was calculated by the formula of the trunk base or the central part or the central part of the trunk base. Based on the expression information of each gene that is significant, the value obtained by the formula of the trunk base and the central part is 1.1 or higher and the value of 0.001 is compared with the xylem side of the central trunk part.
  • the difference in expression between the xylem of the trunk trunk and the xylem of the trunk trunk is 1.5 times or more.
  • the above gene group (SEQ ID NO: 33) is included.
  • the 18 gene groups include gene groups (SEQ ID NOs: 1 to 6, 14, 16, 17, 19) that have a difference in expression between the xylem of the trunk trunk and the xylem of the trunk trunk of 1.5 times or more. -22, 25, 28, 30), gene group (SEQ ID NO: 1 to 6, 14, 16, 17, 22, 25, 30), and a group of genes (SEQ ID NOs: 3, 5, 25, 30) that have an expression differential power of at least twice that between the xylem of the trunk base and the xylem of the central trunk.
  • the six gene groups have gene groups (SEQ ID NOs: 7, 10, 18, 26, 29) whose expression difference on the xylem side is more than double in the middle of the trunk, compared to the phloem side, In the center of the trunk, the gene group (SEQ ID NO: 18, 26, 29) whose expression difference on the xylem side is 2.5 times or more compared to the phloem side is included.
  • the 34 gene groups are expressed in the following (1) to (3) from the detailed expression pattern in the trunk base of the fifth-grade Camaldorensis species or the middle of the trunk and the fifth-grade Groplus species. Broadly classified (Tables 3 and 4).
  • the six gene groups include genes that have a difference in expression of eucalyptus chamaldrensis and eucalyptus globulus stems that are more than 4 times different in the xylem compared to the phloem.
  • the numerical value of the average fluorescence intensity obtained by the experiment is calculated based on the xylem formula of the trunk (base or central part) of the Eucalyptus genus Camaldrensis Z Eucalyptus globulus trunk (base) xylem Z Eucalyptus chamaldorensis trunk (base or middle) xylem calculation.
  • the number of genes obtained by the expression of the xylem of the eucalyptus chamaldrensis tree and the eucalyptus globulus trunk Z As described above, P ⁇ 0.001 is significantly different from ⁇ the gene group in which the expression is increased on the xylem side of the Eucalyptus globulis tree compared to the xylem side of the Eucalyptus camaldrensis ''.
  • the average intensity is almost the same (the fluorescence intensity of the tree part of Eucalyptus genus Camaldrensis relative to the tree part of Eucalyptus globulus trunk is less than l.0 to ll times or P> 0.001)
  • ⁇ Yu -It was classified as a gene group with the same expression on the xylem side of the trunk of the genus Cali genus Camaldrensis and on the xylem side of the stem of the Eucalyptus globules.
  • the seven gene groups show that the difference in the expression on the xylem side is 2.5 times or more in the trunk of Eucalyptus genus Camaldrensis, or in the trunk of Eucalyptus globulus compared with the xylem side.
  • Gene groups (SEQ ID NOs: 8, 13, 19, 24, 25, 33) that have an expression difference of 2.5 times or more on the cervical side, in the trunk of the Eucalyptus genus Camaldrensis, on the xylem side compared to the phloem side Gene group (SEQ ID NOs: 13, 19, 25) Eucalyptus Kamal whose expression difference is 4 times or more, or in Eucalyptus globulus trunks, the expression difference on the phloem side is 4 times or more compared to the xylem side In the trunk of Drensis, the expression difference on the xylem side is more than 10 times compared to the phloem side, or the expression difference on the phloem side compared to the xylem side, A gene group (SEQ ID NO: 25) that is 10 times or more is included.
  • the 13 gene groups include gene groups (SEQ ID NOs: 1-6, 9, 14) that have a three-fold or more differential expression on the xylem side compared to the phloem side in the trunk of the Eucalyptus genus Camaldrensis.
  • Eucalyptus chamaldrensis trunks the gene group is more than double the expression difference on the xylem side compared to the phloem side (SEQ ID NO: 1, 3-6, 14) 16),
  • Eucalyptus genus Camaldrensis trunks include a group of genes (SEQ ID NOs: 1, 3, 5) whose expression difference on the xylem side is more than 10 times that of the phloem side It is.
  • Eucalyptus BAC genomic library was prepared according to the method of J. Agricultural Genomics 5 (http: ⁇ www.ncgr.orc / research / jag) by D. Peterson et al.
  • the prepared library was sorted into 120 384 well plates for each individual clone. From these plates, DNA was prepared for each plate, each column and each row of all plates, and used as a DNA pool for 3D screening by PCR.
  • the genomic clones of each gene were screened by the method.
  • the insert sequence of the BAC clone was determined by the method shown in Example 1. Using the obtained base sequence, the genomic region of each gene was determined based on the cDNA sequence (the cDNA self-sequences described in SEQ ID NOs: 3, 6, 11, 14, 19, 23, 24, 25 were also used). And the determined genome sequences are shown in SEQ ID NOs: 85 to 92, respectively). For clones that were full-length cDNA sequences including the translation initiation codon ATG (which encodes methionine in amino acids), the promoter region was determined based on that sequence. The determined promoter sequence is shown in SEQ ID NOs: 93-100.
  • the TATA-box sequence was determined by analyzing the sequence upstream from the 5 ′ end base of the full-length cDNA using the genetic information processing software “GENETYX” (Software Development Co., Ltd.). It is well known that the promoter region of higher eukaryotes has a TATA-box sequence usually about 10 to 25 bases upstream from the transcription start site! If the position of the determined TATA-bo X is close to the full-length cDNA 5 'terminal base (approx. LOObp), it is judged that cDNA is almost full length and that the upstream side of the terminal base is the promoter region. did. The length of the promoter was set to a maximum of 3 kb upstream from the 5′-terminal base of cDNA with reference to the maximum length (about 3 kb) known in model plants.
  • positions 1 to 660 are the amino acid coding region (SEQ ID NO: 35).
  • the 1st to 602nd positions are the amino acid coding region (SEQ ID NO: 36).
  • the 337th to 3273th positions are the amino acid code region (SEQ ID NO: 37).
  • positions 1 to 1407 are the amino acid coding region (SEQ ID NO: 38).
  • the 1st to 465th positions are the amino acid coding region (SEQ ID NO: 39).
  • the 87th to 3221th positions are the amino acid coding region (SEQ ID NO: 40).
  • the 1st to 699th positions are the amino acid coding region (SEQ ID NO: 41).
  • positions 1 to 1407 are the amino acid coding region (SEQ ID NO: 42).
  • positions 1 to 1122 are the amino acid coding region (SEQ ID NO: 43).
  • the 1st to 1005th positions are the amino acid coding region (SEQ ID NO: 44).
  • positions 313 to 1305 are the amino acid code region (SEQ ID NO: 45).
  • positions 1 to 465 are the amino acid coding region (SEQ ID NO: 46).
  • positions 1 to 762 are the amino acid coding region (SEQ ID NO: 47).
  • positions 375 to 1442 are the amino acid code region (SEQ ID NO: 48).
  • positions 1 to 774 are the amino acid coding region (SEQ ID NO: 49).
  • positions 1 to 498 are the amino acid coding region (SEQ ID NO: 50).
  • positions 68 to 1165 are the amino acid code region (SEQ ID NO: 51).
  • the 117th to 857th positions are the amino acid code region (SEQ ID NO: 52).
  • positions 84 to 968 are the amino acid coding region (SEQ ID NO: 53).
  • positions 1 to 207 are the amino acid coding region (SEQ ID NO: 54).
  • positions 56 to 223 are the amino acid coding region (SEQ ID NO: 55).
  • positions 1 to 621 are the amino acid coding region (SEQ ID NO: 56).
  • the 323rd to 1330th positions are the amino acid code region (SEQ ID NO: 57).
  • positions 240 to 1847 are the amino acid code region (SEQ ID NO: 58).
  • the 273rd to 1928th positions are the amino acid code region (SEQ ID NO: 59).
  • the 1st to 1134th positions are the amino acid coding region (SEQ ID NO: 60).
  • the 1st to 525th positions are the amino acid coding region (SEQ ID NO: 61).
  • the 1st to 1116th positions are the amino acid coding region (SEQ ID NO: 62).
  • the 1st to 195th positions are the amino acid coding region (SEQ ID NO: 63).
  • the first to 138th positions are the amino acid coding region (SEQ ID NO: 64).
  • positions 1 to 564 are the amino acid coding region (SEQ ID NO: 65).
  • the 1st to 711st positions are the amino acid coding region (SEQ ID NO: 66).
  • the 84th to 569th positions are the amino acid coding region (SEQ ID NO: 67).
  • the 1st to 249th positions are the amino acid coding region (SEQ ID NO: 68).
  • the 1st to 2013th positions of the nucleotide sequence represented by SEQ ID NO: 85 are the promoter region (SEQ ID NO: 93), 2350th to 2527th, 3105th to 3181th, 3371th to 3rd. 3422, 3539-3680, 4295-4561, 4924-5269, 5412-5549, 5793-5918, 6001-6213 6649th to 6907th, 6993th to 7192th, 7431th to 7784th, 8069th to 8650th are Etason in the code region, 2528th to 3104th, 3182-3370, 3423-3538, 3681-4294, 4562-4923, 5270-5411, 5550-5792, 5919 -6th, 6214th to 6648th, 6908th to 6992th, 7193th to 7430th, 7785th to 8068th are intron regions.
  • the 1st to 2920th positions of the nucleotide sequence represented by SEQ ID NO: 86 are the promoter region (SEQ ID NO: 94), 3068th to 3139th positions, 3941th to 4141th positions, 4275th to 4275th positions. 4396, 4488-4554, 4670-4808, 5291-5903, 6000-6137, 6426-6551, 6792-7004 Nos. 7091 to 7606, 7998 to 8348, 8695 to 9276 are Etasons in the code area, 3140 to 3940, 4142 to 4274.
  • the 1st to 2700th positions of the nucleotide sequence represented by SEQ ID NO: 87 are the promoter region (SEQ ID NO: 95). Nos. 3013 to 3415, 3508 to 3667, 4610 to 5039 are etasons in the coding region, 3416 to 3507, The 3668th to 4609th positions are intron regions.
  • the 1st to 2707th positions of the base sequence represented by SEQ ID NO: 88 are the promoter region (SEQ ID NO: 96), 3082th to 3170th position, 3365th to 3478th position, 3565th to 3rd position No. 3792, No. 4389 to No. 4828, No. 5124 to No. 5320 are Exons in the code area, No. 3171 to No. 3364, No. 3479 to No. 3564, No. 3793 to No. 4388 4829th to 5123rd position S intron region.
  • the 1st to 2835th positions of the nucleotide sequence represented by SEQ ID NO: 89 are the promoter region (SEQ ID NO: 97), the 2920th to 3011th positions, the 3142th to 3221th positions, the 3345th to 3rd positions.
  • 3489, 3643-3774, 4333-4623 are Exons in the code area, 3012-3141, 3222-3344, 3490-3642 , 3775th position-4332rd position force S intron region.
  • the 1st to 2469th positions of the nucleotide sequence represented by SEQ ID NO: 90 are the promoter region (SEQ ID NO: 98), 2792th to 2924th position, 3027th to 3181th position, 3850th to 3rd position.
  • No. 4035, No. 4202 to No. 4554, No. 5504 to No. 5684 are Exons in the code area, No. 2925 to No. 3026, No. 3182 to No. 3849, No. 4036 to No. 4201 , Positions 4555 to 5503 are S intron regions.
  • positions 1 to 1242 are the promoter region (SEQ ID NO: 99), positions 1482 to 2347, and positions 2452 to 3193 are exons in the coding region. 2nd to 2451th positions are intron regions.
  • the 1st to 2997th positions of the base sequence represented by SEQ ID NO: 92 are the promoter region (SEQ ID NO: 100), the 3270th to 3335th positions, the 3606th position to the 3757th position, the 403th position to the 403th position.
  • No. 4278, No. 4562 to No. 4690, No. 4797 to No. 5732, No. 5844 to No. 5971 are Sae Kwong in the code area, No. 3336 to No. 3605, No. 3758 to No. 4033 , 4279th to 4561th, 4691th to 4796th, 5733th to 584th are the intron regions.
  • Example 5 Selection of transcription factors Transcription factor genes involved in gene expression control (JL Riechmann et al., SCIENCE VOL 290 15 DECEM BER 2000, Kazuo Iwasaki, Kazuo Shinozaki, dynamism of plant genome function, using the annotation information that also obtained the database power shown in Example 1 ) And clones with high homology were searched. As a result, 71 clones were identified. However, these clones also contain transcription factors that are involved in the formation of scab tissue, etc., which is not limited to the formation of wood fibers.
  • RNA extraction method described in Example 1 the present inventors extracted total RNA from the stem base and trunk center of a 5-year-old Camaldrensis species and the trunk base of a Groplus species. At this time, the husk side (the phloem) and the wood fiber forming tissue side (the xylem) of each plant were used. From the total RNA obtained from each individual, mRNA was purified by PolyATract mRNA Isolation System manufactured by Promega. A total of 4 types of mRNA thus obtained were labeled with 2 types of fluorescent dyes (cy3.cy5), respectively, and used as probes in the oligo microarray analysis shown in Example 1 for hybridization. ( Figures 1-3).
  • the hybridization method including the label followed the analysis protocol presented by Agilent Technologies. Fluorescence intensity is calculated from the scanned image, analyzed with Rosetta analysis software (Luminator Ver. 1.0), and all repeated experiments are integrated with a statistical reliability of 99.999%. Information on the expression of 71 clones selected in the trunk base of the drenches species and the center of the trunk, and in the trunk base of the groprus species was obtained.
  • the transcription factor group involved in eucalyptus tree fiber formation is expected to be significantly more strongly expressed on the wood fiber forming tissue side (xylem) than on the crust skin side (phloem).
  • xylem wood fiber forming tissue side
  • phloem crust skin side
  • 11 clones identified a group of genes involved in eucalyptus tree fiber cell wall formation (Table 5- 8).
  • a DNA encoding a protein consisting of the amino acid sequence described in any one of 112 to 122 has the following structural features (domains).
  • amino acid sequence set forth in SEQ ID NO: 112 is a CBF domain, a HAP2 domain,
  • amino acid sequence set forth in SEQ ID NO: 113 is a homeodomain
  • amino acid sequence set forth in SEQ ID NO: 114 is a Coprinusjnating domain
  • amino acid sequence set forth in SEQ ID NO: 115 is a LIM domain
  • amino acid sequence set forth in SEQ ID NO: 116 is a bZIP domain
  • amino acid sequence set forth in SEQ ID NO: 117 is SANT domain, Myb DNA binding domain, RE B1 domain, main,
  • amino acid sequence set forth in SEQ ID NO: 118 is the WRKY domain
  • amino acid sequence described in SEQ ID NO: 119 is homeodomain, leucine zipper domain, the amino acid sequence described in SEQ ID NO: 120 is UPF0023 domain, leucine zipper domain, the amino acid sequence described in SEQ ID NO: 121 is SANT domain, Myb DNA binding domain , RE B1 de, main
  • amino acid sequence set forth in SEQ ID NO: 122 is the Sina domain.
  • the Sequence ID corresponds to SEQ ID NO: 101 to L I 1 in order from the top.
  • the microarray experiment 5y-cam5 X vs P is an experiment comparing the xylem and phloem of the fifth grade eucalyptus 'Camardrensis trunk
  • 5y-caml X vs P is the fifth grade eucalyptus' camaldrenci
  • 5y-glo X vs P represents an experiment comparing the xylem and phloem of the 5th grade Eucalyptus base.
  • the experimental results are expressed as a positive multiple when the former is strong, and as a negative multiple when strong on the husk side, comparing the wood fiber-forming tissue side and the husk side.
  • the five gene groups include a gene group (SEQ ID NO: 102, which has an expression differential power of 3 ⁇ 4 times or more on the xylem side compared to the phloem side in either the trunk base or the trunk central part. 105, 106), a group of genes (SEQ ID NO: 105, 106) whose expression difference on the xylem side is 4 times or more compared to the phloem side in either the trunk base or the trunk central part.
  • Gene group whose expression is increased on the xylem side of the trunk trunk ” is significantly or the same as the average intensity (the fluorescence intensity ratio of the xylem at the center and the xylem at the base is 1.0). Less than -1.1 times or P> 0.001) was classified as a “gene group with similar expression on the xylem side of the trunk base and the xylem side of the trunk trunk”.
  • the two gene groups include a gene group (SEQ ID NOs: 102 and 103) having an expression differential power of ⁇ 3 times in the xylem of the trunk base and the xylem of the trunk central part.
  • the five gene groups have a gene group (SEQ ID NOs: 107, 109 to 111) in which the expression difference on the xylem side is 1.5 times or more in the central trunk portion compared to the phloem side, ⁇ In the center of the trunk, teacher A gene group (SEQ ID NOs: 107 and 109) having an expression differential power of ⁇ times or more on the xylem side compared to the xylem side is included.
  • the value of the average fluorescence intensity obtained from the experiment was calculated based on the trunk of the Eucalyptus chamaldrensis trunk (base or center) Z the trunk of the Eucalyptus globulus (base). It was calculated by the formula of the part or the xylem of the Eucalyptus globulus trunk (base) Z of the trunk of the eucalyptus chamaldrensis (base or middle part).
  • Some genes are not significant or have almost the same average intensity as "a group of genes whose expression increases on the xylem side of the Eucalyptus globulus tree compared to the xylem side of the Eucalyptus genus Camaldrensis" (The fluorescence intensity ratio of the tree part of Eucalyptus genus Camaldrensis compared to the tree part of Eucalyptus globulis
  • the expression difference on the xylem side was more than 4-fold in the trunk of Eucalyptus genus Camaldrensis, or the xylem side in the trunk of Eucalyptus globules
  • the gene (SEQ ID NO: 106) whose expression difference on the phloem side is 4 times or more is included.
  • the Eucalyptus BAC genomic library was screened by the method shown in Example 4. Oligonucleotide primer of SEQ ID NO: 123-144 synthesized based on cDNA sequence information in a DNA pool for 3D screening by PCR, based on cDNA sequence information (odd number of SEQ ID NO: 123-144 is forward primer, even number is reverse) Using primers, the genomic clones of each gene were screened by PCR. [0208] The insert sequence of the BAC clone was determined by the method shown in Example 1.
  • the genomic region and the promoter region of each gene were determined based on the cDNA sequence (SEQ ID NO: 101 to:
  • the genomic sequence determined based on the cDNA sequence described in L11 was determined. , Respectively, as shown in SEQ ID NOs: 145-155).
  • the promoter region was determined based on the full-length cDNA sequence including the translation initiation codon ATG (which encodes methionine in amino acids).
  • the determined promoter sequence is shown in SEQ ID NOs: 156-166.
  • the sequence upstream from the 5 'terminal base of the full-length cDNA was analyzed by genetic information processing software "GENETYX" (Software Development Co., Ltd.), and the TATA-box sequence was determined.
  • the promoter region of higher eukaryotes has a TATA-box sequence usually about 10 to 25 bases upstream from the transcription start site.
  • TATA-box sequence usually about 10 to 25 bases upstream from the transcription start site.
  • nucleotide sequence represented by SEQ ID NO: 101 the 87th to 662nd positions are the amino acid code region (SEQ ID NO: 112).
  • positions 262 to 1188 are the amino acid code region (SEQ ID NO: 113).
  • nucleotide sequence represented by SEQ ID NO: 103 the 257th to 1549th positions are the amino acid code region (SEQ ID NO: 114).
  • positions 159 to 722 are the amino acid code region (SEQ ID NO: 115).
  • positions 101 to 1444 are the amino acid code region (SEQ ID NO: 116).
  • the 163rd to 927th positions are the amino acid code region (SEQ ID NO: 117).
  • the 48th to 2300th positions are the amino acid code region (SEQ ID NO: 118).
  • the 23rd to 778th positions are the amino acid code region (SEQ ID NO: 119).
  • positions 40 to 1101 are the amino acid code region (SEQ ID NO: 120).
  • the 128th to 790th positions are the amino acid code region (SEQ ID NO: 121).
  • positions 269 to 1234 are the amino acid code region (SEQ ID NO: 122).
  • the 1st to 2680th positions of the nucleotide sequence represented by SEQ ID NO: 145 are the promoter region (SEQ ID NO: 156), the 2767th to 2796th positions, the 2884th to 2949th positions, and the 2nd 984th position.
  • -3142, 3265-3585 are etasons in the code area, 29797-2883, 2950-2983, 3143-3264, intron It is.
  • the 1st to 3000th positions of the nucleotide sequence represented by SEQ ID NO: 146 are the promoter region (SEQ ID NO: 157), positions 3262 to 3606, positions 3920 to 4040, and positions 5436. -5626th, 5829th-5960th, 6246th-6383th, Ekson in the force S code area, 3607th-3919th, 4041st-5435th, 5627th ⁇ No. 5828, No. 5961 ⁇ No. 6245 position S intron region.
  • the 1st to 1569th positions of the nucleotide sequence represented by SEQ ID NO: 147 are the promoter region (SEQ ID NO: 158), the 1826th to 2533th positions, the 4847th to 4967th positions, and the 5439th position.
  • -5632, 5709-5840, 5936-6072, 6158 are Etason in the code region, 2534-4846, 4968-5438, Intron regions are 5633 to 5708, 5841 to 5935, and 6073 to 6157.
  • the first to 2979th position of the nucleotide sequence represented by SEQ ID NO: 148 is the promoter region (SEQ ID NO: 159), 3241th to 3375th position, 3967th to 4063th position, 4159th position.
  • -No. 4202, No. 4293-No. 4282, No. 4539-No. 4736 are Eksons in the code area, No. 3376-No. 3966, No. 4064-No. 4158, No. 4203 Positions-4292, 4283-4538 positions S intron region.
  • the 1st to 1288th positions of the nucleotide sequence represented by SEQ ID NO: 149 are the promoter region (SEQ ID NO: 160), 1409th to 1964th position, 3755th to 3959th position, 4th 510th position -4585th, 4564th-4809th, 5066th-5446th Exon in the power code area, 1965-3754, 3960-4509, 4586-4586
  • the 4563rd and 4810th to 5065th positions are intron regions.
  • positions 1 to 1536 are the promoter region (SEQ ID NO: 161), positions 1723 to 1985, and positions 2217 to 2718 are in the code region. No. 1986 to No. 2216 are intron regions.
  • the 1st to 1658th positions of the nucleotide sequence represented by SEQ ID NO: 151 are the promoter region (SEQ ID NO: 162), the 1706th to 2167th positions, the 2407th to 2589th positions, the 2767th position.
  • -No. 3647, No. 3747-No. 3905, No. 4108-No. 4675 are Eksons in the code area, No. 2168-No. 2406, No. 2590-No. 2766, No. 3648-No.
  • the intron region is at positions 3746 and 3906-4107.
  • the 1st to 1979th positions of the nucleotide sequence represented by SEQ ID NO: 152 are the promoter region (SEQ ID NO: 163), positions 2002 to 2427, positions 2714 to 2793, positions 2984.
  • ⁇ Position 3233 is Xun Xun in the coding region
  • Positions 2428 to 2713 and Positions 2794 to 2983 are intron regions.
  • the 1st to 596th positions of the nucleotide sequence represented by SEQ ID NO: 153 are the promoter region (SEQ ID NO: 164), positions 637 to 770, positions 1143 to 1272, positions 1393 to 1 No. 1593, No. 2042 to No. 2209, No. 2939 to No. 3367 are exons in the code region, No. 771 to No. 1142, No. 1273 to No. 1392, No. 1594 to No. 2041 , Positions 2210 to 2938 are intron regions.
  • positions 1 to 2973 are the promoter region (SEQ ID NO: 165), positions 3101 to 3221, positions 3339 to 3468, and positions 4324.
  • ⁇ 4735 is the etason in the coding region, 3222 to 3338, and 3469 to 4323 is the intron region.
  • Sequence number 1 to position 1783 of the nucleotide sequence represented by SEQ ID NO: 155 is the promoter region Region (SEQ ID NO: 166), positions 2052 to 2270, positions 3339 to 3725, positions 4266 to 4625 are Xeon in the coding region, positions 2271 to 3338 , Positions 3726 to 4265 are intron regions.
  • the genes (SEQ ID NOs: 1-34, 101-: L11) expressed in the xylem forming tissues shown in Examples 3 and 6 are regulated in the xylem by their respective promoters. . Therefore, each promoter region can naturally be used for expression control in the xylem of an arbitrary gene. Therefore, reporter genes were connected downstream of these promoters and introduced into plants to confirm expression activity in xylem. In this example, the activity of the promoter region (SEQ ID NO: 93) of the cellulose synthase gene (SEQ ID NO: 3) was analyzed by the particle gun method.
  • the GUS gene was connected as a reporter downstream of the promoter region of the cellulose synthase gene (SEQ ID NO: 3) and introduced into Eucalyptus. Specifically, using the oligonucleotide primer of SEQ ID NO: 167, 168 synthesized based on the genome sequence information described in SEQ ID NO: 85, the BAC clone DNA is in a saddle shape and 5 ′ untranslated region by PCR. The promoter region containing was amplified, and after blunting, it was subcloned upstream of the GUS gene of the binary vector pBI121 vector (Bevan, M., Nucleic Acids Res. 12, 8711-8721, 1984) excluding the 35S promoter.
  • the binary vector pBI121 vector Bevan, M., Nucleic Acids Res. 12, 8711-8721, 1984
  • the above-described plasmid for introduction was introduced into a xylem-forming tissue with a particle gun, and the activity of the GUS gene was analyzed. 5th year eucalyptus growing vigorously Peeling the bark of the trunk and exposing it to the other side, the xylem formation tissue (Fig. 4A) force also cuts the xylem block (Fig. 4B), and the target for particle gun introduction did.
  • the introduction plasmid was coated on gold particles as described below. Immediately mix 50 ⁇ l of plasmid DNA with 51 (0.5 g / ⁇ 1) and 50 ⁇ l of 2.5 2 CaCa2, and add 20 ⁇ l of 0.1M spermidine. To precipitate gold particles.
  • the promoter sequence of the gene described in SEQ ID NOs: 1-34, 101-: L11, in which expression was observed in the xylem by the microarray experiment is the xylem in any gene. It can be used for expression control.
  • Transcription factor is controlled on / off ⁇ by binding to the promoter region of the gene under its control (JL Riechmann et al., SCIENCE VOL 290 15 DECEMBER 2000, Masaaki Iwabuchi, Kazuo Shinozaki, Plant genome function dynamism). Therefore, by analyzing the presence or absence of interaction (binding) with the promoter region of a gene, the relationship between control and control can be understood.
  • the transcription factor described in SEQ ID NO: 101-: L 11 (SEQ ID NO: 112-122) that controls the formation of tree cell walls and the tree described in SEQ ID NO: 1-34 (SEQ ID NO: 35-68) was analyzed the interaction of genes involved in cell wall formation with promoters.
  • the MYB transcription factor protein of SEQ ID NO: 117 and the homeodomain (HD-ZIP) transcription factor protein of SEQ ID NO: 119 are used as promoter regions (sequences of the gene involved in cellulose synthesis (CesA).
  • No. 93) shows the result of interaction between the lignin synthesis gene (CAD, OMT, C4H) promoter (SEQ ID NO: 96, 97, 99, respectively).
  • the MYB cDNA (SEQ ID NO: 106) was PCR-enhanced in a saddle shape using the primers shown in SEQ ID NOs: 169 and 170. Treat the amplified fragment with restriction enzymes Ndel and smal and insert it into pIVEX 1.3 vector (Roche) The reaction was performed at 24 ° C. for 24 hours using a cell-free translation system (wheat germ protein synthesis system kit RTS100; Roche). The reaction mixture (lul) was electrophoresed on SDS-PAGE, and expression of the target protein was confirmed by Western blotting using an ant His antibody.
  • HD-ZIP cDNA (SEQ ID NO: 108) was subjected to PCR using the primers described in SEQ ID NOs: 171 and 172.
  • the amplified DNA fragment was treated with restriction enzymes EcoRV and Sail, inserted into pET32a vector (Novagen), and transformed into E. coli BL21 strain.
  • pET32a vector Novagen
  • each probe was designed with both adjacent probe sequences and a 5 bp overlap, so a total of 40 probe pairs (sense / antisense) were artificially synthesized per promoter. At this time, the sense side DNA is
  • a (fluorescent dye) labeled For the analysis of the interaction, double-stranded DNA in which sense'antisense strands were used was used. For annealing, TAMRA-labeled sense oligo DNA (SEQ ID NO: 173 to 332) and unlabeled antisense oligo DNA (SEQ ID NO: 333 to 492) were adjusted to luM with 0.1M NaCl / TE (pH 8.0). Then, after denaturation treatment at 95 ° C for 5 minutes, it was performed while gradually returning to room temperature over 1 hour. Next, to remove unreacted single-stranded oligo DNA, add 10 mM MgCl and Exonuclease I, react at 37 ° C for 1 hour, and install the MERmaid SPIN Kit (Qbiogene).
  • the sense'antisense strand corresponds to the complementary strand of SEQ ID NO: 173 with SEQ ID NO: 333 (Probe No. 1), and thereafter SEQ ID NO: 174 and 334 (Probe No. 2) to SEQ ID NO: 332 Corresponding in order like 492 (Probe No.160).
  • the DNA-protein binding reaction was performed in 30 ul of reaction solution (25 mM Hepes / KOH (pH 7.9), 50 The reaction was performed with mM KC1, 0.5 mM DTT 5% glycerol, 2 mg / ml BSA 0.05 mg / ml poly (d to dC) '(d to dC), protein lul, 3 nM probe) at room temperature for 20 minutes.
  • FCS Fluorescence Correlation Spectroscopy
  • FCS Fluorescence Correlation Spectroscopy
  • standard Dye was measured 10 times X 5 times and reaction solution was measured 15 times X 5 times.
  • the translation time was calculated from this measured value, and the presence or absence of binding was verified. If binding is observed, the molecular weight of the labeled probe will increase, resulting in a longer translation time than the control.
  • the measured value of the empty vector expressed and each probe added to the reaction solution was used.
  • Fig. 5 shows the results of interaction analysis between the two transcription factors and the four promoters. From the results of the control experiment, it can be concluded that there is an interaction (bonding) when there is a translation time extension of at least 20%. From the results of analysis, in MYB, two sites of CesA promoter (SEQ ID NO: 181 and 341 double-stranded DNA, 199 and 359 double-stranded DNA), one CAD promoter (SEQ ID NO: 246 and 406), OMT promoter 1 (SEQ ID NOs: 274 and 434) and 2 sites of the C4H promoter (SEQ ID NOs: 296 and 456, 326 and 486).
  • CesA promoter SEQ ID NO: 181 and 341 double-stranded DNA, 199 and 359 double-stranded DNA
  • one CAD promoter SEQ ID NO: 246 and 406
  • OMT promoter 1 SEQ ID NOs: 274 and 434
  • 296 and 456, 326 and 486 296 and 456, 326
  • each of the cDNAs (SEQ ID NOs: 106 and 108) was subjected to PCR using the primers described in SEQ ID NOs: 169 to 172.
  • PBI121 vector (Bevan, M., Nucleic Acids Res. 12, 8711-8721, 1984) where the end of the amplified fragment was phosphorylated and blunt ended, and the GUS gene portion was removed beforehand using restriction enzymes. This was ligated downstream of the 35S plug motor and used as an introduction construct.
  • Agrobataterum LBA4404 strain
  • transformants were selected using LB agar medium (25 g / ml each of kanamycin, no, and idaromomycin, 1.0% Agar).
  • LB agar medium 25 g / ml each of kanamycin, no, and idaromomycin, 1.0% Agar.
  • Single colonies were picked from the transformed agrobataterum plates and cultured in LB liquid medium containing antibiotics (kanamycin, no, idaromomycin each g / m) (28.5 ° C, 240 rpm) o culture Thereafter, the agrobacterium was collected by centrifugation (5 ° C., 8000 rpm 5 min), the supernatant was removed, and MS liquid medium was added to gently suspend the precipitate. The suspension was collected in a 50 ml tube and diluted with MS liquid medium so that the OD550 of the suspension was 0.5. This suspension was used for the infection procedure.
  • FIG. 6 shows a transformed tobacco plant overexpressing the MYB transcription factor gene of SEQ ID NO: 106 and the homeodomain (HD-ZIP) transcription factor gene of SEQ ID NO: 108.
  • lignin content and fiber length were measured.
  • Lignin quantification total amount of acid-soluble 'insoluble lignin was determined by the Klason method (Wood Chemistry Experiments II. Chemistry P150-155, Patzlaff et al., 2003, Plant Journal). al., 1999, Kenaf Pro operties, Processing and Products. P149-167).
  • the ratio of lignin per bone dry weight averaged 14.7 ⁇ 1.8% (average of 5 individuals) compared to 19.3 ⁇ 1.1% of the control (wild strain).
  • the fiber length was 560.6 ⁇ 50.9um for the control, whereas it was 620.3 ⁇ 42.lum for the MYB-introduced individuals.
  • a decrease in the lignin ratio relative to the absolute dry weight indicates a relative increase in the cellulose ratio. Therefore, by overexpressing the MYB transcription factor, it was possible to modify the material with a long fiber length with a large amount of cellulose. This indicates that if the expression of this transcription factor is suppressed using a technique such as RNAi, the material can be altered to the opposite material (short fiber length with less cellulose).
  • Cell wall components, conduit distribution density, fiber length, etc. which are the main elements of wood fiber properties, are known as broad-leaved trees. .
  • This fluctuation phenomenon of wood fiber properties due to ground clearance has long been known as vertical fluctuation of the material in the trunk. Comparing each ground height (base ⁇ middle, crown), cellulose increases in cell wall components, and lignin and hemicellulose decrease in the center (above the chest and less than crown). .
  • morphological observation shows that the fiber length with a small conduit distribution density increases by about 20%.
  • the cell wall thickness increases from the crown to the base.
  • the bulk weight of the 5th-year eucalyptus plant was 432 kg / m 2 , the pulp yield was 43.8%, and the fiber length was 0.66 mm.
  • the central part the bulk weight was 525 kg / m 2 , the pulp yield was 53.4%, and the fiber length was 0.86 mm. From these results, it is confirmed in the sample of this example that the vertical fluctuation force is generally called. It was. In addition, it was confirmed that the center part having high fiber weight and high pulp yield and long fibers was superior as the material.
  • the materials of the test samples were examined.
  • the center of trunk of 2 individuals (Sl, 2) of 7th grade Eucalyptus maldorensis was used.
  • RNA extraction and microarray experiments were performed in Examples 1 to 3, and material analysis was performed according to the method described above.
  • the sample site to be examined can be at the trunk base, at the center, or anywhere else.
  • trunk center it is generally expected that the part will have the best material in the individual, so it can be said that the identified material is the best material in the individual.
  • trunk bases The opposite is true for trunk bases.
  • the expression was strong in caml (bad material, one), the gene group was S1, the expression was strong in cam5 (good material, V), and the gene group was highly expressed in S2. From this, it is determined that S2 has a better material than S1. (3) In the inspection of (4), S2 was judged to be closer to the center than S1, and the result was that S2 was superior in material quality. The result of direct comparison between the xylem was also the result that supported it.
  • S1 and S2 were analyzed by the method described in Example 11 (1).
  • S2 in test weight is S 1, 45.7% pulp yield in SI, 50.1% in S2, 0.69 mm fiber length by S1, was 0.75mm in S2 . Therefore, as expected based on the above-mentioned inspection results, S1 was a material close to the base, and S2 was a basic but somewhat central material. Also, comparing both, S2 was relatively better as expected. This As described above, the material of the sample to be tested can be discriminated alive by comparison based on the expression profile shown in the present application.
  • the cellulose of the Globules species generally increases cellulose, while lignin and hemicellulose decrease.
  • morphological observation shows an increase in fiber length when the conduit density is low.
  • the cell wall is thick. The details are unknown, but the nature of the wood fiber-forming tissue differs depending on the species, and it is thought that the difference in the nature of the wood fiber formed is caused.
  • the above-mentioned conventional method for refining kraft pulp using the trunk base of Eucalyptus genus Camaldrensis and the trunk base of Eucalyptus globulus are used.
  • the bulk weight was 432 kg / m 2
  • the pulp yield was 43.8%
  • the fiber length was 0.66 mm.
  • the gross base of Groplus had a bulk weight of 574 kg / m 2 , a pulp yield of 57.1%, and a fiber length of 0.95 mm. From these results, it is generally said that the variability between species varies. It was also confirmed in the sample. In addition, it was also confirmed that the GLOPLUS trunk base with high bulk weight and long fibers was superior as the material.
  • the material By comparing the expression profile of each sample obtained with the taxa in Tables 3 and 7, and examining whether it is similar to the profile of chamaldrensis or that of globus, The material (camaldrensis or glopuls) can be distinguished.
  • the sample site to be examined may be the trunk base, the center, or any other location.
  • Profile of 9 genes showing inverse correlation: 8 genes with camaldrense-like profile (X> P) and 1 gene with globulous-like profile (X P P) Camaldrensis-specific There are 14 genes that show X> P: 12 genes that are chamaldrensic (X> P) and 2 genes that are Globnores (X P).
  • C1 and G1 were analyzed by the method described in Example 11 (1). As a result, 534kg / m 2 at 449kg / m 2, G1 in test weight is C 1, 45.7% for C1 pulp yield 53.6% for G1, fiber length was 0.86mm in C1 0.69 mm, in G1 . Therefore, the material is based on the above inspection results As expected, CI was a camaldrensis-like material and G1 was a globus-like material. Also, comparing both, G1 was relatively better as expected. In this way, the species and material of the test sample can be discriminated alive by comparison based on the expression profile shown in the present application.
  • the present inventors Using the prepared eucalyptus EST database, the present inventors extracted a group of genes involved in cell wall synthesis in a eucalyptus tree fiber-forming tissue by microarray analysis, and the difference in the height and species of the ground. Expression variation was examined. As a result, we identified a cell wall synthetic gene group that is expressed / excited in eucalyptus tree fiber-forming tissues, and a gene group whose expression fluctuates predominantly due to differences in the height and species of the ground. Furthermore, these promoter DNAs were obtained and confirmed to function as promoters for xylem expression. The above gene group and expression information 'promoter DNA can be used to control cell wall synthesis and morphogenesis in wood fiber-forming tissues.
  • the present inventors examined the expression variation due to the difference in the height and species of the ground using the prepared eucalyptus EST database to extract a group of transcription factors involved in tree fiber formation by microarray analysis. .
  • a group of transcription factors involved in eucalyptus tree fiber formation identified a group of genes whose expression fluctuated predominantly depending on the height of the ground, and obtained their promoter DNA.
  • plant materials can be altered by introducing these transcription factors.
  • the above gene set and expression information 'promoter DNA can be used to control wood fiber formation (material).
  • FIG. 1 A graph showing the gene expression intensity of the 5-year-old ginger stem base of Eucalyptus camaldorensis. Probes for oligo microarray analysis by labeling each cRNA, which is synthesized from two types of mRNA extracted from the phloem side of the trunk base and the xylem side, in a cage shape with two fluorescent dyes (cy3.cy5) each And used for hybridization. The fluorescence intensity is calculated from the image obtained by scanning and analyzed with Rosetta's analysis software (Luminator Ver. 1.0), and all repeated experiments are integrated with a statistical reliability of 99.999%. The relative intensity of expression of major gene groups at the base is shown.
  • + (black) in the vicinity of or above the upper part of the two calibration curves shows a significant increase in expression at the wood fiber formation site.
  • + (Light gray) in the middle of the two calibration curves shows a significant decrease in expression, and the two calibration curves are near or below the lower line + (dark gray) Indicates that there is no change in expression.
  • + (black) in the vicinity of or above the upper two of the two calibration curves shows a significant increase in expression at the wood fiber formation site, and is almost in the middle of the two calibration curves
  • + (dark gray) in the vicinity of or below the lower line of the two calibration curves indicates no change in expression.
  • ⁇ 3 It is a figure showing the gene expression intensity of Eucalyptus globulis spp.
  • Each cRNA synthesized from the phloem side and xylem side of the trunk is synthesized in a cage shape and labeled with two fluorescent dyes (cy3.cy5) for use as probes in oligo microarray analysis. I did a hybridization. Image power obtained by scanning Fluorescence intensity is calculated and analyzed with Rosetta analysis software (Luminator Ver. 1.0), and all repeated experiments are integrated with a statistical reliability of 99.999%.
  • FIG. 4 is a graph showing the promoter activity of genes involved in wood fiber formation.
  • A shows the trunk from which the xylem-forming tissue block that is the target for particle gun introduction was excised.
  • B shows the formation of xylem by histological GUS assembly after gene transfer by particle gun. Indicates an organization block.
  • C and D are enlarged photographs of the blue spot where the GUS gene was expressed (the central part surrounded by a circle).
  • FIG. 5-a is a diagram showing the interaction (binding) between the promoter of the cell wall-forming gene group involved in tree fiber formation and the transcription factor group.
  • Each graph shows the analysis results of the MYB transcription factor and the four promoters.
  • the horizontal axis represents the number of the double-stranded DNA probe
  • the vertical axis represents the percentage increase in translation time relative to the control (probe and empty vector reaction solution).
  • the smaller probe number on the horizontal axis of each graph is the upstream region on the 5 ′ side of the promoter, and the larger one is closer to the transcription start point and the downstream region on the 3 ′ side of the promoter.
  • * Indicates a probe that has been recognized to interact.
  • FIG. 5-b is a diagram showing the interaction (binding) between the promoter of the cell wall-forming gene group involved in tree fiber formation and the transcription factor group.
  • Each graph shows the results of analysis of HD-ZIP transcription factors and four promoters.
  • the horizontal axis represents the number of the double-stranded DNA probe
  • the vertical axis represents the rate of increase in translation time relative to the control (probe and empty vector reaction solution) in%.
  • the smaller probe number on the horizontal axis of each graph is the upstream region on the 5 ′ side of the promoter, and the larger probe number is closer to the transcription start point and the downstream region on the 3 ′ side of the promoter.
  • the asterisk (*) indicates a probe that has been recognized to interact!
  • [6] It is a diagram showing a transformed tobacco plant into which a transcription factor has been introduced.
  • A is a transformant introduced with a MYB transcription factor
  • B is a transformant introduced with a homeodomain transcription factor.

Abstract

By constructing and using a eucalyptus EST database, genes participating in cell wall synthesis in a eucalyptus cellulosic fiber-forming tissue and transcriptional factors regulating the expression of these genes are extracted by the microarray analysis and differences in the expression thereof are analyzed depending on aboveground height and species. As a result, cell wall synthesis genes expressed in the eucalyptus cellulosic fiber-forming tissue and transcriptional factors are specified and it is identified that they show dominantly coordinated variation in the expression depending on aboveground height and species. Moreover, promoter DNAs of them are obtained. It is considered that the above-described genes and expression data/promoter DNAs are usable in controlling the cell wall synthesis by the cellulosic fiber-forming tissue and morphological formation.

Description

明 細 書  Specification
植物樹幹の木繊維細胞壁を形成 ·制御する機能を有するタンパク質をコ ードする DNAとそのプロモーター DNA  DNA that codes for the protein that functions to form and control the tree fiber cell walls of plant trunks and its promoter DNA
技術分野  Technical field
[0001] 本発明は、植物樹幹の木繊維細胞壁を形成する機能を有するタンパク質をコード する DNA、そのプロモーター DNA、並びにその利用に関する。  [0001] The present invention relates to DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk, a promoter DNA thereof, and use thereof.
本発明はまた、植物の転写因子をコードする DNA、プロモーター DNA断片、並びに その利用に関する。  The present invention also relates to DNA encoding plant transcription factors, promoter DNA fragments, and uses thereof.
背景技術  Background art
[0002] 人類は長い間、榭幹 ·根，葉 ·枝等の木質バイオマスを、製紙、建築、飼料、燃料等 の多岐にわたる産業分野で利用してきた。世界の木質バイオマス消費量は毎年増加 傾向にあり、用途別にみると、薪炭用材の消費が過半数を占め、開発途上地域では 今でも増加傾向を示している。製材、製紙用の木材チップなどの産業用材も、先進 地域の消費量は若干の減少に転じたものの、開発途上国地域における消費量は増 カロしている。先進地域では文明の発達とともに、森林の農地への転用や資材としての 利用等により森林を大きく減少させてきたが、最近植林活動によってわず力ながら増 カロしている。しかし、開発途上地域では、かっては先進国の商業伐採により、近年は 人口増加にともなって、生活燃料や農地の需要が拡大し、世界全体における森林面 積が急激に減少しつづけている。こうしたことから、二酸化炭素固定化能の低下によ る地球温暖化などの問題が挙げられるようにもなつてきた。これらの問題を解決しつ つ、近年、産業的に製材、製紙用木材チップの安定供給などを目指した植林事業が 世界各地で盛んに行われて 、る。  [0002] Mankind has long used woody biomass such as trunks, roots, leaves, and branches in a wide variety of industrial fields such as papermaking, construction, feed, and fuel. The world's woody biomass consumption is increasing every year, and by use, the consumption of wood-burning wood accounts for the majority, and it is still increasing in developing regions. Consumption of industrial materials such as lumber and wood chips for papermaking has also been increasing in developed regions, although consumption in developed regions has slightly decreased. In advanced areas, along with the development of civilization, forests have been greatly reduced by diverting forests to farmland and using them as materials. However, in developing regions, due to commercial logging in developed countries, the demand for fuel and farmland has increased in recent years as the population has increased, and the forest area throughout the world has been rapidly decreasing. As a result, problems such as global warming due to a decline in carbon dioxide fixation ability have been raised. While solving these problems, in recent years, afforestation projects aiming at a stable supply of lumber and wood chips for industrial use have been actively carried out around the world.
[0003] 木質バイオマスを利用した産業は、将来的にも、持続的利用可能な資源を活用で きるとして地球環境問題改善の観点から再認識されており、現行の化石資源に替わ るカーボンソースの利用による循環型産業として期待されて 、る。具体的事例を示す と、我が国の製紙会社では、原料である木質バイオマスの持続的かつ安定的確保達 成のため、ユーカリ属、アカシア属等の熱帯早生榭を中心とした植林事業を精力的 に進めている。その植林事業規模の例として、王子製紙では 2010年度までに 30万 ha の植林面積を目指し、オセアニア、東南アジアなどの環太平洋を中心に広い地域で 行っている。このことは、事業レベルでの大規模植林を行うことによるバイオマスのェ 業利用、再生と!/、う循環型の物質生産 (バイオマスのリサイクル)を他の産業分野に 先駆けて進めて!/、ることに他ならな!、。 [0003] Industries that use woody biomass have been re-recognized from the perspective of improving global environmental issues as they can continue to use sustainable resources in the future, and carbon sources to replace current fossil resources It is expected as a recycling industry by use. To give concrete examples, Japanese paper companies are actively engaged in afforestation projects centering on early tropical pods such as Eucalyptus and Acacia in order to achieve a sustainable and stable supply of woody biomass as a raw material. We are proceeding to. As an example of the scale of the afforestation project, Oji Paper is aiming for an afforestation area of 300,000 ha by fiscal 2010, and is conducting it in a wide area centering on the Pacific Rim such as Oceania and Southeast Asia. This means that biomass is used and regenerated through large-scale plantation at the business level, and recycling material production (biomass recycling) is ahead of other industrial fields! /, Other than that!
[0004] さらに、このような植林事業の進展にあわせ、主要な木質バイオマスである樹幹に おける木繊維形成を人為的に制御することで、その構成要素である細胞壁の成分量 (セルロース ·へミセルロース ·リグニン)並びに質を可変すること、さらに繊維形態 (木 繊維細胞の伸長）を自由に可変することが可能となれば、木質バイオマスの量的な 増大並びに質的な改良が見込まれ、これによつて将来のエネルギー利用や工業原 料としての用途拡張も見込まれる。 [0004] Furthermore, along with the progress of such afforestation projects, artificially controlling the formation of tree fibers in the trunk, which is the main woody biomass, the amount of cell wall components (cellulose hemi If it becomes possible to change the quality of cellulose and lignin) as well as the fiber morphology (elongation of wood fiber cells) freely, it is expected that the amount of woody biomass will increase and the quality will be improved. As a result, future energy use and application expansion as industrial raw materials are also expected.
[0005] 榭幹における木質バイオマスの大部分は木繊維と呼ばれる組織細胞で構成されて V、る。木繊維組織の比重 ·細胞壁構造や成分 (セルロース ·へミセルロース ·リグニン） 等は、木繊維の性質を決める主要要素であり、工業原材料として利用する場合、各 製品仕様にあった多様な性質が求められる。例えば、高い比重の木繊維から製造し た紙は、低い比重のそれと比較して、叩解に対する抵抗性、引裂き強さなどは高いが 、反面、パルプシートの密度、引張り、破裂および耐折強度などは低い。  [0005] Most of the woody biomass in the trunk is composed of tissue cells called wood fibers. The specific gravity of the wood fiber structure, cell wall structure and components (cellulose, hemicellulose, lignin), etc. are the main factors that determine the properties of wood fibers. When used as industrial raw materials, there are various properties that meet the product specifications. Desired. For example, paper made from wood fibers with high specific gravity has higher resistance to beating and tear strength than those with low specific gravity, but on the other hand, density of pulp sheet, tensile, burst and folding strength, etc. Is low.
[0006] この木繊維細胞の性質は、広葉樹 '針葉樹を問わず、地上高 *榭齢'種により変動 することが知られている (非特許文献 1、非特許文献 2)。榭齢による木繊維性質の変 動現象につ 、ては古くから成熟材 ·未成熟材として、また地上高による変動現象につ いては材質の榭幹内垂直変動として、そして種による変動現象については榭種間変 動として知られる。例えば、成熟材と未成熟材を相互比較すると、成熟材では細胞壁 成分については、セルロースが増大し、リグニン、へミセルロースが減少する。また、 形態的観察では、導管分布密度が少なぐ繊維長は増加が認められる。同様の違い 力 地上高や種の違い (非特許文献 1〜3)でも発生する。詳細は不明であるが、各 木繊維形成組織での細胞壁合成が異なるため、形成される木繊維性質の差が発生 して 、るものと考えられて 、る。このような異なる性質の細胞壁合成を担う各遺伝子群 の特定はなされていない。 [0007] 近年、草本を中心としたモデル植物等を用いたゲノムレベルでの遺伝子解析研究 が盛んに行われている。草本では、特定時期に特定器官において、わずかな木繊維 しか形成しない。例えば、主要なモデルであるシロイヌナズナの場合、栄養成長期に は木繊維はほとんど形成されず、生殖成長期の花茎内のごく一部に形成される（非 特許文献 4)。榭木の繊維細胞は、形成層組織から分化し木繊維 (wood fiber)と呼ば れるのに対し、草本の繊維細胞は主として維管束と維管束の間の柔細胞力 分ィ匕し 、束間繊維（interfascicular fiber)と呼ばれる。このように、草本の繊維細胞は、多年 にわたつて肥大成長する木本の木繊維細胞とは同一でない。したがって、真に木繊 維形成に必要な遺伝子群を同定するためには、榭木を用いて解析しなければならな い。 [0006] It is known that the nature of this wood fiber cell varies depending on the species of broad-leaved tree, 'coniferous tree, regardless of whether it is high above the age * dawn' (Non-Patent Document 1, Non-Patent Document 2). About the change phenomenon of the wood fiber property by the age, it has been a mature and immature wood for a long time, the fluctuation phenomenon due to the ground height is the vertical fluctuation of the material in the trunk, and the fluctuation phenomenon by the species Is known as inter-species variation. For example, when mature and immature wood are compared with each other, in mature wood, cellulose increases in cell wall components, while lignin and hemicellulose decrease. In addition, morphological observation shows an increase in fiber length when the conduit density is low. Similar difference Forces also occur at ground clearance and species differences (Non-Patent Documents 1 to 3). Although details are unknown, it is thought that the difference in the properties of the wood fibers formed is caused by the difference in cell wall synthesis in each wood fiber-forming tissue. The gene groups responsible for cell wall synthesis with such different properties have not been identified. [0007] In recent years, gene analysis studies at the genome level using model plants such as herbs have been actively conducted. In herbs, only a few wood fibers are formed in specific organs at specific times. For example, in the case of Arabidopsis thaliana, which is a major model, almost no wood fibers are formed during the vegetative growth period, but only a small part of the flower stems during the reproductive growth period (Non-patent Document 4). Fibrous fiber cells differentiate from the formation layer tissue and are called wood fibers, whereas herbaceous fiber cells mainly divide the parenchyma force between the vascular bundles, and the inter-bundle fibers ( It is called interfascicular fiber). Thus, herb fiber cells are not the same as wood fiber cells that grow over many years. Therefore, in order to identify a gene group that is really necessary for the formation of wood fibers, it must be analyzed by using an oak tree.
[0008] 植物の木繊維形成関連遺伝子群やその制御因子は、単に既知の制御因子との相 同性検索やモチーフ検索では区別'同定できない。例えば、木繊維細胞壁の主成分 であるセルロースやリグニンの合成経路には多数の酵素が関わっており、それらの遺 伝子は植物ゲノム中で大きなファミリーを形成している（非特許文献 5)。さらにファミリ 一内では、一次細胞壁の合成に関わるものや二次細胞壁の合成に関わるものなど があり、機能する場所や役割が様々に分担されている。配列モチーフや相同性検索 力 では、単に高い相同性が示されるだけで、役割等を特定することはできない。例 えば、セルロース合成に関わる酵素 cellulose synthase (CesA)は、シロイヌナズナゲ ノム中に 40個程度予想されて!、る。その中で一次壁のセルロース合成に必須な Ces Aとして RSW1 (非特許文献 6)が同定されており、二次壁セルロース合成に必須な Ce sAとして IRX3 (非特許文献 7)が同定されている。両者のアミノ酸配列の相同性は、全 体で 65%であり部分的には 90%を超え大変高い。このように、単純に配列の相同性 スコアだけでは役割を同定できないため、木繊維細胞壁合成遺伝子群を特定するた めには、詳細な発現解析等が必要である。  [0008] Plant fiber formation-related genes and their regulatory factors cannot be distinguished and identified simply by homology search or motif search with known regulatory factors. For example, many enzymes are involved in the synthesis pathway of cellulose and lignin, which are the main components of wood fiber cell walls, and these genes form a large family in the plant genome (Non-patent Document 5). Furthermore, within the family 1, there are those related to the synthesis of the primary cell wall and those related to the synthesis of the secondary cell wall, and the functioning places and roles are divided in various ways. Sequence motifs and homology search capabilities simply indicate high homology and cannot identify roles. For example, about 40 cellulose synthases (CesA) involved in cellulose synthesis are expected in Arabidopsis thaliana! Among them, RSW1 (Non-patent document 6) has been identified as Ces A essential for cellulose synthesis of the primary wall, and IRX3 (Non-patent document 7) has been identified as Ce sA essential for the synthesis of secondary wall cellulose. . The homology between the amino acid sequences of both is 65% overall and is very high, exceeding 90% in part. Thus, since the role cannot be identified simply by the sequence homology score, detailed expression analysis and the like are necessary in order to identify the wood fiber cell wall synthesis genes.
[0009] セルロース 'リグニン合成系の遺伝子力 ゲノム中に多数のファミリーを形成し、多 様なレベルの群に分かれて発現しているのと同様に、それらの制御因子もまた多数 のファミリーを形成し多様なレベルの群に分かれて発現しており、配列上のモチーフ や相同性検索だけでは役割を区別できない。例えば、シロイヌナズナゲノム中の制 御因子は 50以上のファミリー、 1500以上の遺伝子が同定されている（非特許文献 8 ) oその中でも、例えば MYBファミリーに属する制御因子は約 190個にもおよび、詳細 な発現解析等によりそれぞれが特有な生命現象に関与していることが少しずつ明ら かにされつつある。また、例えば、木繊維に含まれるリグニンに代表されるフエ-ルプ ロパノイド生合成系の制御因子としてタバコで同定された制御因子 NtLIMl (特許文 献 1)と相同なものを、公知の検索サイト（http://www.ncbi.nlm.nih.gov/blast/)にて検 索すると、多数の LIMタンパク質と高い相同性を示す。木繊維を形成しないラットの LI Mタンパク質とさえ、高い相同性スコアでヒットする。シロイヌナズナでは、少なくとも 10 個以上の LIMタンパク質と高いスコア（56〜87%)でヒットするが、その中でどの LIM タンパク質がシロイヌナズナのリグニン合成系の制御に関わっているか不明である。 このように、制御因子の役割は既知の制御因子との相同性だけでは同定できないた め、詳細な発現解析等を行わなければならない。し力しながら、榭木における制御因 子の網羅的な発現解析の知見はな 、。 [0009] Gene power of cellulose 'lignin synthesis system Just as many families are formed in the genome and expressed in various levels, their regulators also form many families They are expressed in groups of various levels, and their roles cannot be distinguished only by sequence motifs or homology searches. For example, the control in the Arabidopsis genome More than 50 families and more than 1500 genes have been identified (Non-patent Document 8). O Among them, there are about 190 regulatory factors belonging to the MYB family. It is gradually becoming clear that it is involved in specific life phenomena. In addition, for example, a homologous search factor NtLIMl (Patent Document 1) identified in tobacco as a regulator of the biopropanoid biosynthesis system represented by lignin contained in wood fiber is known ( Searching at http://www.ncbi.nlm.nih.gov/blast/) shows high homology with many LIM proteins. Even rat LI M proteins that do not form wood fibers are hit with a high homology score. In Arabidopsis, at least 10 LIM proteins and a high score (56-87%) are hit, but it is unclear which LIM protein is involved in the control of the lignin synthesis system in Arabidopsis. Thus, since the role of a regulatory factor cannot be identified only by homology with known regulatory factors, detailed expression analysis and the like must be performed. However, there is no knowledge of comprehensive expression analysis of regulatory factors in Kashiwagi.
[0010] 前述のように、草本では木本と同一の木繊維は形成されない。また、単に既知の遺 伝子との相同性だけで、木繊維細胞壁のセルロース ·リグニン合成関連遺伝子群を 特定することはできない。つまり、草本の繊維形成に関わる情報を、そのまま榭木の 木繊維形成に利用することはできな ヽ。真に木繊維細胞壁合成に関わるセルロース 'リグニン合成系の遺伝子を特定するのは、草本では困難である。榭木を用いて木繊 維形成部位の発現解析を行うことで、木繊維細胞壁のセルロース ·リグニン合成に必 要な遺伝子群を同定できる。し力しながら、榭木を用いて木繊維細胞壁合成に関わ るセルロース ·リグニン合成系の遺伝子の特定を行った例は少な ヽ（非特許文献 9と 1 0)。 [0010] As described above, the same wood fiber as the wood is not formed in the herb. In addition, it is not possible to identify a group of genes related to cellulose / lignin synthesis in the wood fiber cell wall simply by homology with known genes. In other words, information related to herbaceous fiber formation cannot be used as it is for the formation of wood fiber. It is difficult to identify the genes of the cellulose lignin synthesis system that are truly involved in the synthesis of wood fiber cell walls. By analyzing the expression of the wood fiber formation site using coconut trees, it is possible to identify the genes required for cellulose / lignin synthesis in the wood fiber cell wall. However, there are few examples in which a cellulosic lignin synthesis system gene involved in wood fiber cell wall synthesis was identified using an oak (Non-patent Documents 9 and 10).
[0011] 草本と木本間における繊維形成時に働く遺伝子群の違いだけでなぐ木本植物に おいても、多様な木繊維細胞壁合成遺伝子群の存在が予想されている。例えば、若 年と壮年では葉や樹幹の形態等が違うことから、年齢によって異なる複数の木繊維 細胞壁合成遺伝子群が機能していることが予想されている。また、前述のように榭高 による垂直変動や、榭種による榭種間変動が生じることから、同一個体内や種間でも 異なる複数の木繊維細胞壁合成遺伝子群が、各々で特有に機能して!/ヽることが予 想される。これらの各木繊維細胞壁合成遺伝子群は、配列上は互いに高い相同性を 示すだけで、どれがどのレベルの木繊維細胞壁合成遺伝群に属するかは区別でき ない。この区別を行うためには、木繊維形成部位での詳細な発現解析が必要である 。し力しながら、そのような知見はない。 [0011] A variety of wood fiber cell wall synthesis genes are expected to exist in woody plants, which are based only on the difference in the genes that act during fiber formation between grass and wood. For example, because the morphology of leaves and trunks is different between young and middle age, it is expected that multiple tree fiber cell wall synthetic genes that function according to age will function. Also, as mentioned above, vertical fluctuations due to drought height and fluctuations among varieties due to varieties occur, so that a plurality of tree fiber cell wall synthetic genes that are different within the same individual or between species function uniquely. ! I think. These tree fiber cell wall synthetic genes only show high homology with each other in sequence, and it is not possible to distinguish which belongs to which level of wood fiber cell wall synthetic genes. In order to make this distinction, detailed expression analysis at the wood fiber formation site is necessary. However, there is no such knowledge.
[0012] 木本植物においても、ポプラやマツを用いてゲノムレベルでの榭木解析研究がなさ れている（非特許文献 7と 9)。しかしながら、用いられている材料は、根や葉'形成層 といった別々の器官や組織単位であり、それらにおける遺伝子発現情報を報告して いるにすぎない。異なる地上高'種の木繊維形成組織での発現解析を行い、多様な レベルの木繊維細胞壁合成に関わる遺伝子群やそれらのプロモーター情報，発現 情報に関するゲノムレベルでの網羅的な知見はない。  [0012] Also in woody plants, research on the analysis of silkworms at the genome level using poplar and pine has been conducted (Non-Patent Documents 7 and 9). However, the materials used are separate organs and tissue units such as roots and leaves' formation layers, and only report gene expression information in them. There is no comprehensive knowledge at the genome level regarding gene groups involved in various levels of wood fiber cell wall synthesis, their promoter information, and expression information by conducting expression analysis in different types of tree fiber-forming tissues on the ground.
[0013] ところで、植林事業の現場では、植林木の生長状態は外見で判断しており、またパ ルプ原料としての質を知るためには、伐採後に化学的な手法でパルプ原料である榭 幹の木繊維の分析 (材質分析)を行っている。生長状態については、経験と勘に頼つ ているため、外見では判別できない栄養分の不足等の問題点は把握できない。そし て、伐採後にしかその材質が分力もないため、生長中の植林木の木繊維形成 (材質 )がどのような状況であるの力、将来どのような木繊維が形成されるのか等はわ力 ず 、良い材質を作り出すための施業 (施肥や間伐等)方針もたたない。また、優良な植 林木を選抜する場合、生長中であれば外見上判断できる生長量 (榭高 ·直径)を指 標にするしかなぐ材質を予測して選抜することができない。このように、榭幹の木繊 維形成 (材質)を生長中に予測することは、植林地での施業や選抜 '育種、質の良い 製紙原料の確保を考える上で大変重要である。  [0013] By the way, at the site of the afforestation project, the growth state of the afforestation tree is judged by appearance, and in order to know the quality as a pulp material, a trunk that is a pulp material by a chemical method after cutting is used. Wood fiber analysis (material analysis). As for the growth state, it relies on experience and intuition, so it cannot grasp problems such as lack of nutrients that cannot be distinguished by appearance. And since the material is only divided after logging, what is the power of tree fiber formation (material) in planted trees during growth, what kind of tree fiber will be formed in the future? There is no policy of working (fertilization, thinning, etc.) to produce good materials. In addition, when selecting a good plantation tree, it is impossible to select a material that can only be judged by using the amount of growth (height and diameter) that can be judged by appearance if it is growing. Thus, predicting trunk fiber formation (material) during growth is very important in consideration of plantation operations, selective breeding, and securing high-quality papermaking raw materials.
[0014] 前述のように、植物は同じ種，同じ個体内でも木繊維形成状態等の生長状態に関 して違いがあり、それは外見上判別できない。そのため、生長中に木繊維形成 (材質 )を予測するための検査マーカーが必要とされてきた。しかしながら、これまでに生長 状態や木繊維形成状態の検査マーカーは開発されていな力つた。  [0014] As described above, even in the same species and in the same plant, there is a difference in the growth state such as the state of tree fiber formation, which cannot be discriminated in appearance. Therefore, there has been a need for an inspection marker for predicting the formation (material) of wood fiber during growth. However, until now, there has been no development of test markers for growth and wood fiber formation.
[0015] なお、本出願の発明に関連する先行技術文献情報を以下に記す。  [0015] Prior art document information related to the invention of the present application is described below.
特許文献 1：特許第 3444191号  Patent Document 1: Patent No. 3444191
非特許文献 1 :島地謙 ·須藤彰司 ·原田浩著「木材の組織」、森北出版、 1976、 111-21 5 Non-Patent Document 1: Ken Shimaji · Akuji Sudo · Hirada Hiroshi "Wood Organization", Morikita Publishing, 1976, 111-21 Five
非特許文献 2 :古野毅'澤辺攻著「木材科学講座 2 ·組織と材質」、海青社、 1994、 109 -137  Non-Patent Document 2: Satoshi Furuno 'Osamu Sawabe's "Wood Science Course 2 Organization and Material", Kaiseisha, 1994, 109 -137
非特許文献 3 : Wood Science and Technology. 2001;35:229-243.  Non-Patent Document 3: Wood Science and Technology. 2001; 35: 229-243.
非特許文献 4 : Plant Physiology, 2001, 126:477-479.  Non-Patent Document 4: Plant Physiology, 2001, 126: 477-479.
非特許文献 5 : Plant Physiology, 2000, 124: 495-498.  Non-Patent Document 5: Plant Physiology, 2000, 124: 495-498.
非特許文献 6 : Science 1998, 279:717-720.  Non-Patent Document 6: Science 1998, 279: 717-720.
非特許文献 7 : Plant Cell 1999, 11:769-780.  Non-Patent Document 7: Plant Cell 1999, 11: 769-780.
非特許文献 8：岩淵雅榭 ·篠崎一雄編「植物ゲノム機能のダイナミズム」シユープリン ガ一'フエアラーク東京、 2001、 1-34  Non-Patent Document 8: Masami Iwabuchi · Kazuo Shinozaki “Dynamics of Plant Genomic Function” Syupurin Gaichi 'Fairark Tokyo, 2001, 1-34
非特許文献 9： GenomeBiol. 2002;3(12):REVIEWS1033.  Non-Patent Document 9: GenomeBiol. 2002; 3 (12): REVIEWS1033.
非特許文献 10 : Proc Natl Acad Sci U S A 2001 Dec 4;98(25): 14732-7.  Non-Patent Document 10: Proc Natl Acad Sci U S A 2001 Dec 4; 98 (25): 14732-7.
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0016] 木質バイオマスの性質は、木繊維の細朐齄及び形餱等によって変動する。この木 繊維の細朐齄及び形態等の形成を人為的に制御するこ で、効果的かつ効率的に 工業原材料としての木質バイオマスの生産を行うことができる。また、世界はいまだ大 量の石油、天然ガスなどの化石資源に依存している。今、新しい技術を持ってこの現 状を変えていくためには、木質バイオマスとしての樹木の効率的な活用こそ力 その 切り札となる。海外諸国においては、榭木バイオマスの有効活用に関する技術確立 は、今世紀初頭における重要課題と位置づけられている。実際、米国では針葉樹の マツ、広葉樹のポプラ、カナダでは同じくトウヒ、ポプラが、北欧ではポプラがその対 象榭種として、国家プロジェクトとしてゲノム科学的な解析により研究を開始して 、る。 地球規模での木質バイオマスのより効果的かつ効率的生産を担うためにも、植物に おける木繊維細胞形成を制御する遺伝子を同定する必要性は非常に高い。  [0016] The nature of woody biomass varies depending on the fineness and shape of wood fibers. By artificially controlling the formation of fine fibers and shapes of the wood fibers, woody biomass can be produced as an industrial raw material effectively and efficiently. In addition, the world still depends on fossil resources such as large quantities of oil and natural gas. Now, in order to change this situation with new technology, efficient utilization of trees as woody biomass is the key to power. In overseas countries, the establishment of technology for effective utilization of Kashiwagi biomass is positioned as an important issue at the beginning of this century. In fact, coniferous pine, broad-leaved poplar in the United States, spruce and poplar in Canada, and poplar in Scandinavia are the target species. In order to contribute to more effective and efficient production of woody biomass on a global scale, it is highly necessary to identify genes that control the formation of wood fiber cells in plants.
[0017] 本発明の課題は、植物樹幹の木繊維細胞壁を形成する機能を有するタンパク質を コードする DNA、植物の転写因子をコードする DNA、そのプロモーター DNA、並びに これらを含むプラスミド、ならびに、そのプラスミドにより形質転換されてなる植物細胞 、微生物もしくは植物体を提供することにある。また、植物樹幹の木繊維細胞壁を形 成する機能を有するタンパク質と植物の転写因子をコードする DNAに関し、植物の 榭幹木部の検査用マーカーとしての用途を提供することも課題とする。 [0017] An object of the present invention is to provide a DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk, a DNA encoding a plant transcription factor, a promoter DNA thereof, a plasmid containing these, and a plasmid thereof Plant cells transformed by To provide microorganisms or plants. Another object of the present invention is to provide a protein having a function of forming a tree fiber cell wall of a plant tree trunk and a DNA encoding a plant transcription factor as a test marker for a plant trunk tree part.
課題を解決するための手段  Means for solving the problem
[0018] 本発明者らは、鋭意研究を重ねた結果、本課題を解決するためには、ゲノム科学 的なアプローチによる、ユーカリ木繊維細胞壁を形成する遺伝子群の取得と、発現- 機能に関する包括的な解析を行うべきとの結論にいたった。すなわち、木繊維形成 組織を含む発現遺伝子データベース (ESTデータベース）を作製し、異なる地上高 · 種の木繊維形成部位における発現解析を行うことで、配列の相同性検索では区別で きない目的の遺伝子群を網羅的に同定 '取得し、それら遺伝子のプロモーター DNA 断片を取得、解析する方法を用いることが望ましいとの結論にいたった。さらに、遺伝 子工学技術を使用した改良遺伝子の人為的発現制御によって、人類が利用する上 で有用な特性を植物に与える為には、新 Uヽ特性が適当な植物組織で選択的に発 現される様々な組織に特異的な遺伝子群の同定が必要であるとの結論に 、たった。  [0018] As a result of intensive research, the present inventors have solved the problem by acquiring a group of genes that form the eucalyptus tree fiber cell wall by a genomic-scientific approach and comprehending expression-function. I came to the conclusion that a realistic analysis should be done. In other words, by creating an expression gene database (EST database) that includes tree fiber-forming tissues and analyzing expression at different tree fiber formation sites on different ground types, the target genes that cannot be distinguished by sequence homology search It was concluded that it would be desirable to use a method of comprehensively identifying groups and obtaining them, and obtaining and analyzing promoter DNA fragments of those genes. Furthermore, in order to give plants useful characteristics for human use by controlling artificial expression of improved genes using genetic engineering technology, new U ヽ characteristics are selectively expressed in appropriate plant tissues. They concluded that it was necessary to identify genes specific to various tissues.
[0019] 本研究の遂行に当たり、ユーカリにおける解析リソースの整備を行い、具体的には 各種遺伝子ライブラリー、榭幹部、葉部、根部の各組織毎に ESTデータベースを作製 した。これにカ卩え、モデル植物として世界中で広く活用されているシロイヌナズナにつ いても、遺伝子ライブラリーや細胞壁生合成に関わる突然変異体を保有すると共に、 これらの原因遺伝子にっ 、ては既に解析済みである。  [0019] In carrying out this study, eucalyptus analysis resources were prepared. Specifically, EST databases were created for various gene libraries, trunks, leaves, and roots. In contrast, Arabidopsis, which is widely used around the world as a model plant, possesses gene libraries and mutants involved in cell wall biosynthesis, and these causative genes have already been used. It has been analyzed.
[0020] 本発明者らは、作製したユーカリ ESTデータベースを用いて、マイクロアレイ解析に よるユーカリ木繊維形成組織において細胞壁合成に関与する遺伝子群の抽出と、そ れらの地上高.種の違いによる発現変動を調べた。その結果、地上高'種の違いで優 位に発現が変動するユーカリ木繊維形成組織の細胞壁合成遺伝子群を特定した。 さらにそれらのプロモーター DNAを取得した。上記遺伝子群とプロモーター DNAは、 木繊維形成組織の細胞壁合成および形態形成の制御や木繊維特異的な遺伝子発 現制御に利用できると考えられる。  [0020] The present inventors use the prepared eucalyptus EST database to extract a group of genes involved in cell wall synthesis in a eucalyptus tree fiber-forming tissue by microarray analysis, and to determine the height of the ground. Expression variation was examined. As a result, we identified a cell wall synthetic gene group of Eucalyptus tree fiber-forming tissue whose expression is preferentially varied depending on the difference between the above-ground species. Furthermore, their promoter DNA was obtained. It is considered that the above gene group and promoter DNA can be used to control cell wall synthesis and morphogenesis of wood fiber-forming tissues and to control gene expression specific to wood fiber.
[0021] 本発明の遺伝子群は、詳細な発現様式から、「ユーカリ属カマルドレンシスの榭幹 基部および榭幹中央部にお 、て、師部側と比較して木部側で発現レベルが上昇し ている遺伝子群」、「ユーカリ属カマルドレンシスの榭幹基部において、師部側と比較 して木部側で発現レベルが減少し、榭幹中央部において、師部側と比較して木部側 で発現レベルが上昇している遺伝子群」、「ユーカリ属カマルドレンシスの榭幹中央 部において、師部側と比較して木部側で発現レベルが上昇し、榭幹基部において、 師部側と木部側で発現レベルが同等な遺伝子群」、「ユーカリ属カマルドレンシスお よびユーカリ属グロブルスの樹幹にぉ 、て、師部側と比較して木部側で発現レベル が上昇している遺伝子群」、「ユーカリ属カマルドレンシスの榭幹において、師部側と 比較して木部側で発現レベルが上昇し、ユーカリ属グロブルスの榭幹において、師 部側と比較して木部側で発現レベルが減少している遺伝子群」、「ユーカリ属カマル ドレンシスの榭幹において、師部側と比較して木部側で発現レベルが上昇し、ユー力 リ属グロブルスの樹幹にぉ ヽて、師部側と木部側で発現レベルが同等な遺伝子群」 の 6グループに大別された。この遺伝子群の発現 (レベル)情報は、植物体の生長状 態や木繊維形成状態、材質の量的'質的な事前予測に利用できると考えられる。こ のことにより、植林事業において、生長状態をもとに適切な施業 (除草，間伐等)を適 切な時期に施すことができ、ひいてはパルプ製造の効率化、コストの削減、新規な特 性を持った紙の製造が可能になる。 [0021] From the detailed expression pattern, the gene group of the present invention has an expression level on the xylem side compared to the phloem side in the trunk base and central trunk of Eucalyptus genus Camaldrensis. Rise In the trunk base of Eucalyptus genus Camaldrensis, the expression level is decreased on the xylem side compared to the phloem side, and in the middle trunk, the xylem is compared with the phloem side. `` Gene group with increased expression level on the side '', `` In the central trunk of Eucalyptus genus Camaldrensis, the expression level increased on the xylem side compared to the phloem side, and at the base of the trunk, Gene groups with the same expression level on the xylem side and xylem side ”,“ the eucalyptus chamaldrensis and eucalyptus globulus trunks have increased expression levels on the xylem side compared to the phloem side. In the trunk of Eucalyptus genus Camaldrensis, the expression level increased on the xylem side compared to the phloem side, and in the trunk of the Eucalyptus globules, the xylem compared to the phloem side. Genes with reduced expression levels on the side " “In the trunk of the Eucalyptus genus Camal Drensis, the expression level increased on the xylem side compared to the phloem side, and the expression level on the phloem side and the xylem side increased compared to the trunk of the Eucalyptus globulus. It was roughly divided into 6 groups of “equivalent genes”. It is considered that the expression (level) information of this gene group can be used for quantitative and qualitative advance prediction of plant growth state, wood fiber formation state, and material. As a result, in the afforestation business, appropriate operations (weeding, thinning, etc.) can be performed at an appropriate time based on the growth state, and as a result, the efficiency of pulp production, cost reduction, and new characteristics can be improved. It becomes possible to manufacture the paper you have.
[0022] 本発明によって得られたユーカリ榭幹の木繊維細胞壁を形成する機能を有するタ ンパク質をコードする DNA、並びにそれらを統括的に制御する技術と発現情報 ·プロ モーター DNAについては、その 1つの成果として、これらの遺伝子群を用いて得られ た遺伝子組換えユーカリ新品種の特性による、高セルロース、低リグニン、細胞壁の 厚いもの、薄いもの、繊維長の長いもの、短いもの等様々な量的かつ質的な変化が 期待される。また、発現情報に基づき地上高、種などの材質や生長状態の判別に使 用することが期待される。  [0022] DNA encoding a protein having a function of forming a eucalyptus trunk tree fiber cell wall obtained by the present invention, as well as techniques for comprehensively controlling them and expression information. As one result, various characteristics such as high cellulose, low lignin, thick cell walls, thin ones, long fiber lengths, short ones, etc. depending on the characteristics of the new transgenic eucalyptus varieties obtained using these genes. Quantitative and qualitative changes are expected. In addition, it is expected to be used for discrimination of ground height, species and other materials and growth status based on expression information.
[0023] 即ち、本発明は、植物樹幹の木繊維細胞壁を形成する機能を有するタンパク質を コードする DNA、そのプロモーター DNA断片、並びにその利用に関し、以下の〔1〕〜 〔18〕を提供するものである。  [0023] That is, the present invention relates to DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk, a promoter DNA fragment thereof, and use thereof, which provide the following [1] to [18] It is.
〔1〕以下の（a)〜（e)のいずれかに記載の植物樹幹の木繊維細胞壁を形成する機 能を有するタンパク質をコードする DNA。 (a)配列番号： 35〜68のいずれかに記載のアミノ酸配列力 なるタンパク質をコード する DNA [1] DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk according to any one of the following (a) to (e). (a) DNA encoding a protein having the amino acid sequence ability described in any of SEQ ID NOs: 35 to 68
(b)配列番号： 1〜34のいずれかに記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 1-34
(c)配列番号： 35〜68のいずれかに記載のアミノ酸配列力 なるタンパク質と 50%以 上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with a protein having an amino acid sequence ability according to SEQ ID NO: 35-68
(d)配列番号： 1〜34のいずれかに記載の塩基配列力 なる DNAとストリンジェントな 条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence according to SEQ ID NO: 1-34
(e)配列番号： 35〜68のいずれかに記載のアミノ酸配列において 1または複数のァ ミノ酸が置換、欠失、付加、および/または挿入されたアミノ酸配列からなるタンパク 質をコードする DNA  (e) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 35-68
〔2〕植物がユーカリである、〔1〕に記載の DNA。  [2] The DNA according to [1], wherein the plant is eucalyptus.
〔3〕〔1〕または〔2〕に記載の DNAの部分 DNA。  [3] A partial DNA of the DNA according to [1] or [2].
〔4〕〔1〕〜〔3〕の 、ずれかに記載の DNAが固定ィ匕された基板。  [4] A substrate on which the DNA according to any one of [1] to [3] is immobilized.
〔5〕植物の榭幹の師部側および木部側において、以下の（a)〜（e)に記載の少なくと も 1つの DNAの発現レベルを検出し、師部側および木部側における該 DNAの発現レ ベルを比較する工程を含む、植物の榭幹木部の生長状態を検査する方法。  [5] At the phloem side and xylem side of the plant trunk, the expression level of at least one DNA described in the following (a) to (e) is detected, and the phloem side and xylem side are detected. A method for examining the growth state of a stem trunk of a plant, comprising a step of comparing the expression levels of the DNA.
(a)配列番号： 35〜64または 66〜68のいずれかに記載のアミノ酸配列からなるタン パク質をコードする DNA  (a) DNA encoding a protein consisting of the amino acid sequence set forth in any of SEQ ID NOs: 35 to 64 or 66 to 68
(b)配列番号： 1〜30または 32〜34のいずれかに記載の塩基配列を含む DNA (b) SEQ ID NO: DNA comprising the nucleotide sequence set forth in any of 1-30 or 32-34
(c)配列番号： 35〜64または 66〜68のいずれかに記載のアミノ酸配列からなるタン パク質と 50%以上の相同性を有するタンパク質をコードする DNA (c) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 64 or 66 to 68
(d)配列番号： 1〜30または 32〜34のいずれかに記載の塩基配列からなる DNAとス トリンジェントな条件下でハイブリダィズする DNA  (d) SEQ ID NO: DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence of any one of 1-30 or 32-34
(e)配列番号： 35〜64または 66〜68のいずれかに記載のアミノ酸配列において 1ま たは複数のアミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配列か らなるタンパク質をコードする DNA  (e) a protein comprising the amino acid sequence of SEQ ID NO: 35-64 or 66-68, wherein one or more amino acids are substituted, deleted, added, and Z or inserted. DNA to encode
〔6〕異なる 2つの榭幹木部において、以下の（a)〜（e)に記載の少なくとも 1つの DNA の発現レベルを検出し、異なる 2つの榭幹木部における該 DNAの発現レベルを比較 する工程を含む、異なる 2つの榭幹木部の生長状態を検査する方法。 [6] The expression level of at least one DNA described in the following (a) to (e) is detected in two different trunk trunks, and the expression level of the DNA in two different trunk trunks is compared. A method for inspecting the growth status of two different trunks.
(a)配列番号： 35〜40、 42、 48、 50、 51、 53〜56、 58、 59、 62、 64、 66、または 6 7のいずれかに記載のアミノ酸配列力 なるタンパク質をコードする DNA  (a) DNA encoding the protein having the amino acid sequence ability according to any one of SEQ ID NOs: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62, 64, 66, or 67
(b)配列番号： 1〜6、 8、 14、 16、 17、 19〜22、 24、 25、 28、 30、 32、または 33の Vヽずれかに記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence of SEQ ID NO: 1-6, 8, 14, 16, 17, 19-22, 24, 25, 28, 30, 32, or 33
(c)配列番号： 35〜40、 42、 48、 50、 51、 53〜56、 58、 59、 62、 64、 66、または 6 (c) SEQ ID NO: 35-40, 42, 48, 50, 51, 53-56, 58, 59, 62, 64, 66, or 6
7の 、ずれかに記載のアミノ酸配列力 なるタンパク質と 50%以上の相同性を有する タンパク質をコードする DNA DNA encoding a protein having 50% or more homology with a protein having the amino acid sequence ability described in 7
(d)配列番号： 1〜6、 8、 14、 16、 17、 19〜22、 24、 25、 28、 30、 32、または 33の いずれかに記載の塩基配列力 なる DNAとストリンジヱントな条件下でノヽイブリダィズ する DNA  (d) SEQ ID NOs: 1 to 6, 8, 14, 16, 17, 19 to 22, 24, 25, 28, 30, 32, or 33 Noisy Breeding DNA
(e)配列番号： 35〜40、 42、 48、 50、 51、 53〜56、 58、 59、 62、 64、 66、または 6 7のいずれかに記載のアミノ酸配列において 1または複数のアミノ酸が置換、欠失、 付加、および Zまたは挿入されたアミノ酸配列力もなるタンパク質をコードする DNA 〔7〕植物の榭幹の師部側および木部側において、以下の（a)〜（e)に記載の少なくと も 1つの DNAの発現レベルを検出し、師部側および木部側における該 DNAの発現レ ベルを比較する工程を含む、植物の榭幹木部の生長状態を検査する方法。  (e) SEQ ID NOs: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62, 64, 66, or 67, wherein one or more amino acids are DNA encoding a protein that also has substitution, deletion, addition, and Z or inserted amino acid sequence ability [7] On the phloem side and xylem side of the plant trunk, as described in (a) to (e) below A method for examining the growth state of a stem xylem of a plant, comprising the step of detecting the expression level of at least one DNA and comparing the expression level of the DNA on the phloem side and the xylem side.
(a)配列番号： 35〜40、 42、 43, 45〜48、 50、 51、 53〜59、 61、 62、 64, 66,ま たは 67のいずれかに記載のアミノ酸配列力 なるタンパク質をコードする DNA (a) SEQ ID NO: 35-40, 42, 43, 45-48, 50, 51, 53-59, 61, 62, 64, 66, or 67 DNA to encode
(b)配列番号： 1〜6、 8、 9、 11〜14、 16、 17、 19〜25、 27、 28、 30、 32、または 3 3の!、ずれかに記載の塩基配列を含む DNA (b) SEQ ID NO: 1 to 6, 8, 9, 11 to 14, 16, 17, 19 to 25, 27, 28, 30, 32, or 33!
(c)配列番号： 35〜40、 42、 43, 45〜48、 50、 51、 53〜59、 61、 62、 64, 66,ま たは 67のいずれかに記載のアミノ酸配列力 なるタンパク質と 50%以上の相同性を 有するタンパク質をコードする DNA  (c) SEQ ID NO: 35 to 40, 42, 43, 45 to 48, 50, 51, 53 to 59, 61, 62, 64, 66, or 67 DNA encoding a protein with 50% or more homology
(d)配列番号： 1〜6、 8、 9、 11〜14、 16、 17、 19〜25、 27、 28、 30、 32、または 3 (d) SEQ ID NO: 1-6, 8, 9, 11-14, 16, 17, 19-25, 27, 28, 30, 32, or 3
3のいずれかに記載の塩基配列力もなる DNAとストリンジヱントな条件下でノヽイブリダ ィズする DNA DNA that also has a base sequence ability as described in any of the above 3 and DNA that is hybridized under stringent conditions
(e)配列番号： 35〜40、 42、 43, 45〜48、 50、 51、 53〜59、 61、 62、 64, 66,ま たは 67のいずれかに記載のアミノ酸配列において 1または複数のアミノ酸が置換、欠 失、付加、および Zまたは挿入されたアミノ酸配列からなるタンパク質をコードする DN A (e) SEQ ID NO: 35-40, 42, 43, 45-48, 50, 51, 53-59, 61, 62, 64, 66, DN A which encodes a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence of any one of 67
〔8〕異なる 2つの榭幹木部において、以下の（a)〜（c)に記載の少なくとも 1つの DNA の発現レベルを検出し、異なる 2つの榭幹木部における該 DNAの発現レベルを比較 する工程を含む、異なる 2つの榭幹木部の生長状態を検査する方法。  [8] The expression level of at least one DNA described in (a) to (c) below is detected in two different trunk trunks, and the expression level of the DNA in two different trunk trunks is compared. A method for inspecting the growth status of two different trunks.
(a)配列番号： 45、 46、 51、 55、または 56のいずれかに記載のアミノ酸配列からなる タンパク質をコードする DNA  (a) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56
(b)配列番号： 11、 12、 17、 21、または 22のいずれかに記載の塩基配列を含む DN (b) DN containing the nucleotide sequence set forth in any of SEQ ID NOS: 11, 12, 17, 21, or 22
A A
(c)配列番号： 45、 46、 51、 55、または 56のいずれかに記載のアミノ酸配列からなる タンパク質と 50%以上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56
(d)配列番号： 11、 12、 17、 21、または 22のいずれかに記載の塩基配列からなる D NAとストリンジェントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 11, 12, 17, 21, or 22
(e)配列番号： 45、 46、 51、 55、または 56のいずれかに記載のアミノ酸配列におい て 1または複数のアミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配 列からなるタンパク質をコードする DNA  (e) consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56 DNA encoding the protein
〔9〕〔1〕または〔2〕に記載の DNAによりコードされるタンパク質。  [9] A protein encoded by the DNA of [1] or [2].
〔10〕以下の（a)〜（c)のいずれかの塩基配列からなるプロモーター DNA。  [10] A promoter DNA comprising the nucleotide sequence of any of the following (a) to (c).
(a)配列番号： 93〜： LOOのいずれかに記載の塩基配列  (a) SEQ ID NO: 93-: The nucleotide sequence according to any one of LOO
(b) (a)の塩基配列において、 1又は数個の塩基が欠失、置換又は付加された塩基 配列からなり、かつ、（a)の DNAからなるプロモーターと実質的に同一のプロモーター としての機能を有する塩基配列  (b) In the base sequence of (a), it consists of a base sequence in which one or several bases are deleted, substituted or added, and is substantially the same promoter as the promoter composed of the DNA of (a). Functional base sequence
(c) (a)の塩基配列の一部を含み、かつ、（a)の DNAからなるプロモーターと実質的 に同一のプロモーターとしての機能を有する塩基配列  (c) a base sequence that includes a part of the base sequence of (a) and has a function as a promoter substantially the same as the promoter comprising the DNA of (a)
〔11〕さらに、植物の木繊維形成組織に特異的発現に関与する転写因子を認識する 塩基配列を機能的に結合させた、〔10〕に記載のプロモーター DNA。  [11] The promoter DNA according to [10], further comprising a base sequence that recognizes a transcription factor involved in specific expression in a tree fiber-forming tissue of a plant.
〔12〕以下の（a)〜（e)のいずれかに記載の DNA。 (a)〔1〕に記載の DNAの転写産物と相補的なアンチセンス RNAをコードする DNA[12] The DNA according to any one of (a) to (e) below. (a) DNA encoding an antisense RNA complementary to the transcription product of DNA according to [1]
(b)〔1〕に記載の DNAの転写産物を特異的に開裂するリボザィム活性を有する RNA をコードする DNA (b) a DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcript of the DNA of [1]
(c) RNAi効果により、〔1〕に記載の DNAの発現を抑制する RNAをコードする DNA (c) DNA encoding RNA that suppresses the expression of DNA according to [1] by RNAi effect
(d)共抑制効果により、〔1〕に記載の DNAの発現を抑制する RNAをコードする DNA(d) a DNA encoding an RNA that suppresses the expression of the DNA according to [1] due to a co-suppression effect;
(e)〔1〕に記載の DNAの転写産物に対してドミナントネガティブな形質を有するタンパ ク質をコードする DNA (e) DNA encoding a protein having a dominant negative trait for the transcript of the DNA described in [1]
〔13〕〔1〕〜〔3〕または〔10〕〜〔12〕のいずれかに記載の DNAを有する組換えべクタ  [13] A recombinant vector comprising the DNA according to any one of [1] to [3] or [10] to [12]
〔14〕〔13〕に記載のベクターを含むプラスミドを保持する微生物。 [14] A microorganism carrying a plasmid comprising the vector according to [13].
〔15〕〔14〕に記載のベクターが導入された形質転換植物細胞。  [15] A transformed plant cell into which the vector according to [14] has been introduced.
〔16〕 [15]に記載の形質転換植物細胞から再分化した形質転換植物体。  [16] A transformed plant regenerated from the transformed plant cell according to [15].
〔17〕〔16〕に記載の形質転換植物体の子孫またはクローンである、形質転換植物体  [17] A transformed plant that is a descendant or clone of the transformed plant according to [16]
〔18〕〔16〕または〔17〕に記載の形質転換植物体の繁殖材料。 [18] A propagation material for the transformed plant according to [16] or [17].
[0024] 本発明者らは、鋭意研究を重ねた結果、本課題を解決するためには、ゲノム科学 的なアプローチによる、ユーカリ木繊維細胞壁を形成する遺伝子群の発現を制御す る転写因子群の取得と、発現'機能に関する包括的な解析を行うべきとの結論にい たった。すなわち、木繊維形成組織を含む発現遺伝子データベース (ESTデータべ ース)を作製し、異なる地上高'種の木繊維形成部位における発現解析を行うことで、 配列の相同性検索では区別できない目的の遺伝子群を網羅的に同定 ·取得し、そ れら遺伝子のプロモーター DNA断片を取得、解析する方法を用いることが望ま 、と の結論にいたった。さらに、遺伝子工学技術を使用した改良遺伝子の人為的発現制 御によって、人類が利用する上で有用な特性を植物に与える為には、新しい特性が 適当な植物組織で選択的に発現される様々な組織に特異的な遺伝子群の同定が 必要であるとの結論に 、たった。 [0024] As a result of intensive research, the present inventors have solved the problem by a group of transcription factors that control the expression of genes that form eucalyptus tree fiber cell walls by a genomic scientific approach. And concluded that a comprehensive analysis of expression 'function should be performed. In other words, by creating an expression gene database (EST database) that includes wood fiber-forming tissues and performing expression analysis at different types of tree fiber formation sites on the ground, it is not possible to distinguish by sequence homology search. It was concluded that it would be desirable to use a method of comprehensively identifying and acquiring gene groups and acquiring and analyzing promoter DNA fragments of these genes. Furthermore, in order to give plants useful characteristics for human use by controlling the artificial expression of improved genes using genetic engineering techniques, various new characteristics can be selectively expressed in appropriate plant tissues. They concluded that it was necessary to identify specific genes for specific tissues.
[0025] 本研究の遂行に当たり、ユーカリにおける解析リソースの整備を行い、具体的には 各種遺伝子ライブラリー、榭幹部、葉部、根部の各組織毎に ESTデータベースを作製 した。これにカ卩え、モデル植物として世界中で広く活用されているシロイヌナズナにつ いても、遺伝子ライブラリーや細胞壁生合成に関わる突然変異体を保有すると共に、 これらの原因遺伝子にっ 、ては既に解析済みである。 [0025] In carrying out this research, analysis resources in Eucalyptus were developed, and specifically, EST databases were created for various gene libraries, trunks, leaves, and roots. did. In contrast, Arabidopsis, which is widely used around the world as a model plant, possesses gene libraries and mutants involved in cell wall biosynthesis, and these causative genes have already been used. It has been analyzed.
[0026] 本発明者らは、作製したユーカリ ESTデータベースを用いて、マイクロアレイ解析に よるユーカリ木繊維細胞壁形成に関与する転写因子群の抽出と、それらの地上高 · 種の違いによる発現変動を調べた。その結果、地上高.種の違いで優位に発現が変 動する、ユーカリ木繊維細胞壁形成遺伝子群の発現を制御する転写因子群を特定 した。さらにそれらのプロモーター DNAを取得した。  [0026] Using the prepared eucalyptus EST database, the present inventors examined the transcriptional factors involved in eucalyptus tree fiber cell wall formation by microarray analysis, and the expression variation due to differences in the height and species of the ground. It was. As a result, we identified a group of transcription factors that regulate the expression of Eucalyptus tree fiber cell wall-forming genes, whose expression changes predominantly depending on the height of the ground. Furthermore, their promoter DNA was obtained.
[0027] 本発明の遺伝子群は、詳細な発現様式から、「ユーカリ属カマルドレンシスの榭幹 基部および榭幹中央部にお 、て、師部側と比較して木部側で発現レベルが上昇し ている遺伝子群」、「ユーカリ属カマルドレンシスの榭幹中央部において、師部側と比 較して木部側で発現レベルが上昇し、榭幹基部において、師部側と木部側で発現レ ベルが同等な遺伝子群」、「ユーカリ属カマルドレンシスおよびユーカリ属グロブルス の榭幹にぉ 、て、師部側と比較して木部側で発現レベルが上昇して 、る遺伝子群」 、「ユーカリ属カマルドレンシスの榭幹において、師部側と比較して木部側で発現レ ベルが上昇し、ユーカリ属グロブルスの榭幹において、師部側と比較して木部側で発 現レベルが減少している遺伝子群」、「ユーカリ属カマルドレンシスの榭幹において、 師部側と比較して木部側で発現レベルが上昇し、ユーカリ属グロブルスの榭幹にお Vヽて、師部側と木部側で発現レベルが同等な遺伝子群」の 5グループに大別された。 この遺伝子群の発現 (レベル)情報は、植物体の生長状態や木繊維形成状態、材質 の量的 ·質的な事前予測に利用できると考えられる。このことにより、植林事業におい て、生長状態をもとに適切な施業 (除草 ·間伐等)を適切な時期に施すことができ、ひ いてはノ ルプ製造の効率化、コストの削減、新規な特性を持った紙の製造が可能に なる。 [0027] From the detailed expression pattern, the gene group of the present invention has an expression level on the xylem side compared to the phloem side in the trunk base and central trunk of the Eucalyptus genus Camaldrensis. In the central trunk of Eucalyptus genus Camaldrensis, the expression level rises on the xylem side compared to the phloem side, and the phloem side and xylem on the trunk base Genes whose expression level is the same on the side of the eucalyptus genus, and that the expression level on the xylem side is higher than that on the phloem side of the eucalyptus genus Camaldrensis and Eucalyptus globules. Group ”,“ In the trunk of Eucalyptus genus Camaldrensis, the expression level increased on the xylem side compared to the phloem side, and in the trunk of Eucalyptus globules, the xylem side compared to the phloem side. Genes whose expression levels are reduced in In Rencis trunk, the expression level is increased on the xylem side compared to the phloem side, and the expression group on the eucalyptus globulus trunk V is equal to the expression level on the phloem side and xylem side. ”Was divided into 5 groups. It is considered that the expression (level) information of this gene group can be used for quantitative and qualitative advance prediction of plant growth state, wood fiber formation state, and material. As a result, in the afforestation project, appropriate operations (weeding, thinning, etc.) can be carried out at the appropriate time based on the growth state. This makes it possible to produce paper with characteristics.
[0028] 本発明によって得られたユーカリの転写因子をコードする DNA、並びにそれらを統 括的に制御する技術と発現情報 'プロモーター DNAについては、その 1つの成果とし て、これらの遺伝子群を用いて得られた遺伝子組換えユーカリ新品種の特性による、 高セルロース、低リグニン、細胞壁の厚いもの、薄いもの、繊維長の長いもの、短いも の等様々な量的かつ質的な変化が期待される。また、発現情報に基づき地上高、種 などの材質や生長状態の判別に使用することが期待される。 [0028] DNA encoding eucalyptus transcription factors obtained by the present invention, as well as technology and expression information for overall control of them. For promoter DNA, these genes are used as one result. High cellulose, low lignin, thick cell walls, thin cells, long fiber lengths, short ones Various quantitative and qualitative changes are expected. In addition, it is expected to be used to distinguish the ground height, species, and other materials and growth status based on the expression information.
即ち、本発明は、植物の転写因子をコードする DNA、そのプロモーター DNA断片、 並びにその利用に関し、以下の〔1〕〜〔17〕を提供するものである。  That is, the present invention provides the following [1] to [17] regarding DNA encoding plant transcription factors, promoter DNA fragments thereof, and use thereof.
〔1〕以下の（a)〜（e)の!、ずれかに記載の植物の転写因子をコードする DNA。 [1] A DNA encoding a plant transcription factor described in any one of (a) to (e) below.
(a)配列番号： 112〜 122の 、ずれかに記載のアミノ酸配列力もなるタンパク質をコ ードする DNA  (a) DNA encoding a protein having an amino acid sequence ability described in any one of SEQ ID NOs: 112 to 122
(b)配列番号：101〜： L 11の!、ずれかに記載の塩基配列を含む DNA  (b) SEQ ID NO: 101-: DNA comprising the base sequence described in L11!
(c)配列番号： 112〜 122の 、ずれかに記載のアミノ酸配列力もなるタンパク質と 50 %以上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein of SEQ ID NO: 112 to 122, which also has an amino acid sequence ability described in any one of
(d)配列番号： 101〜111のいずれかに記載の塩基配列からなる DNAとストリンジ ントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 101-111
(e)配列番号： 112〜 122の 、ずれかに記載のアミノ酸配列にお 、て 1または複数の アミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配列力 なるタンパ ク質をコードする DNA  (e) In the amino acid sequence according to any one of SEQ ID NOS: 112 to 122, one or a plurality of amino acids are substituted, deleted, added, and encoded by a protein having an amino acid sequence ability by Z or insertion DNA
〔2〕植物がユーカリである、〔1〕に記載の DNA。  [2] The DNA according to [1], wherein the plant is eucalyptus.
〔3〕〔1〕または〔2〕に記載の DNAの部分 DNA。  [3] A partial DNA of the DNA according to [1] or [2].
〔4〕〔1〕〜〔3〕の 、ずれかに記載の DNAが固定ィ匕された基板。  [4] A substrate on which the DNA according to any one of [1] to [3] is immobilized.
〔5〕植物の榭幹の師部側および木部側において、以下の（a)〜（e)に記載の少なくと も 1つの DNAの発現レベルを検出し、師部側および木部側における該 DNAの発現レ ベルを比較する工程を含む、植物の榭幹木部の生長状態を検査する方法。  [5] At the phloem side and xylem side of the plant trunk, the expression level of at least one DNA described in the following (a) to (e) is detected, and the phloem side and xylem side are detected. A method for examining the growth state of a stem trunk of a plant, comprising a step of comparing the expression levels of the DNA.
(a)配列番号： 112〜 118または 120〜 122の!、ずれかに記載のアミノ酸配列からな るタンパク質をコードする DNA  (a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 112-118 or 120-122
(b)配列番号： 101〜107または 109〜111のいずれかに記載の塩基配列を含む D NA  (b) SEQ ID NO: 101-107 or 109-111
(c)配列番号： 112〜 118または 120〜 122の!、ずれかに記載のアミノ酸配列からな るタンパク質と 50%以上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with a protein consisting of the amino acid sequence of SEQ ID NO: 112-118 or 120-122
(d)配列番号： 101〜107または 109〜111のいずれかに記載の塩基配列からなる DNAとストリンジェントな条件下でハイブリダィズする DNA (d) SEQ ID NO: consisting of the base sequence described in any one of 101 to 107 or 109 to 111 DNA that hybridizes with DNA under stringent conditions
(e)配列番号： 112〜 118または 120〜 122の!ヽずれかに記載のアミノ酸配列にお いて 1または複数のアミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸 配列からなるタンパク質をコードする DNA  (e) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence of SEQ ID NO: 112-118 or 120-122. DNA encoding
〔6〕異なる 2つの榭幹木部において、以下の（a)〜（e)に記載の少なくとも 1つの DNA の発現レベルを検出し、異なる 2つの榭幹木部における該 DNAの発現レベルを比較 する工程を含む、異なる 2つの榭幹木部の生長状態を検査する方法。  [6] The expression level of at least one DNA described in the following (a) to (e) is detected in two different trunk trunks, and the expression level of the DNA in two different trunk trunks is compared. A method for inspecting the growth status of two different trunks.
(a)配列番号： 113または 114に記載のアミノ酸配列力もなるタンパク質をコードする DNA  (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 113 or 114
(b)配列番号： 102または 103に記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 102 or 103
(c)配列番号： 113または 114に記載のアミノ酸配列力もなるタンパク質と 50%以上 の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in SEQ ID NO: 113 or 114
(d)配列番号： 102または 103に記載の塩基配列からなる DNAとストリンジ ントな条 件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 102 or 103
(e)配列番号： 113または 114に記載のアミノ酸配列にお 、て 1または複数のアミノ酸 が置換、欠失、付加、および/または挿入されたアミノ酸配列力 なるタンパク質をコ ードする DNA  (e) DNA encoding a protein having an amino acid sequence ability in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence set forth in SEQ ID NO: 113 or 114
〔7〕植物の榭幹の師部側および木部側において、以下の（a)〜（e)に記載の少なくと も 1つの DNAの発現レベルを検出し、師部側および木部側における該 DNAの発現レ ベルを比較する工程を含む、植物の榭幹木部の生長状態を検査する方法。  [7] At the phloem side and xylem side of the plant trunk, the expression level of at least one DNA described in the following (a) to (e) is detected, and the phloem side and xylem side are detected. A method for examining the growth state of a stem trunk of a plant, comprising a step of comparing the expression levels of the DNA.
(a)配列番号： 113〜 117の 、ずれかに記載のアミノ酸配列力 なるタンパク質をコ ードする DNA  (a) DNA encoding a protein having the amino acid sequence ability described in any one of SEQ ID NOS: 113 to 117
(b)配列番号： 102〜106のいずれかに記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence according to any one of SEQ ID NOs: 102 to 106
(c)配列番号： 113〜 117の 、ずれかに記載のアミノ酸配列力もなるタンパク質と 50 %以上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein of SEQ ID NO: 113 to 117 which also has an amino acid sequence ability described in any
(d)配列番号： 102〜 106のいずれかに記載の塩基配列からなる DNAとストリンジェ ントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 102 to 106
(e)配列番号： 113〜 117の 、ずれかに記載のアミノ酸配列にお 、て 1または複数の アミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配列力 なるタンパ ク質をコードする DNA (e) one or more of the amino acid sequences according to any one of SEQ ID NOS: 113 to 117 DNA encoding a protein with amino acid sequence ability with amino acid substitutions, deletions, additions, and Z or insertions
〔8〕異なる 2つの榭幹木部において、以下の（a)〜（c)に記載の少なくとも 1つの DNA の発現レベルを検出し、異なる 2つの榭幹木部における該 DNAの発現レベルを比較 する工程を含む、異なる 2つの榭幹木部の生長状態を検査する方法。  [8] The expression level of at least one DNA described in (a) to (c) below is detected in two different trunk trunks, and the expression level of the DNA in two different trunk trunks is compared. A method for inspecting the growth status of two different trunks.
(a)配列番号： 116に記載のアミノ酸配列力もなるタンパク質をコードする DNA (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 116
(b)配列番号： 105に記載の塩基配列を含む DNA (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 105
(c)配列番号： 116に記載のアミノ酸配列力もなるタンパク質と 50%以上の相同性を 有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in SEQ ID NO: 116
(d)配列番号： 105に記載の塩基配列からなる DNAとストリンジ ントな条件下でノ、ィ ブリダィズする DNA  (d) DNA that is hybridized under stringent conditions with DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 105
(e)配列番号： 116に記載のアミノ酸配列において 1または複数のアミノ酸が置換、欠 失、付加、および Zまたは挿入されたアミノ酸配列からなるタンパク質をコードする DN A  (e) DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence set forth in SEQ ID NO: 116
〔9〕〔1〕または〔2〕に記載の DNAによりコードされるタンパク質。  [9] A protein encoded by the DNA of [1] or [2].
〔10〕以下の（a)〜（c)のいずれかの塩基配列からなるプロモーター DNA。  [10] A promoter DNA comprising the nucleotide sequence of any of the following (a) to (c).
(a)配列番号：156〜166の!、ずれかに記載の塩基配列  (a) SEQ ID NO: 156 to 166!
(b) (a)の塩基配列において、 1又は数個の塩基が欠失、置換又は付加された塩基 配列からなり、かつ、（a)の DNAからなるプロモーターと実質的に同一のプロモーター としての機能を有する塩基配列  (b) In the base sequence of (a), it consists of a base sequence in which one or several bases are deleted, substituted or added, and is substantially the same promoter as the promoter composed of the DNA of (a). Functional nucleotide sequence
(c) (a)の塩基配列の一部を含み、かつ、（a)の DNAからなるプロモーターと実質的 に同一のプロモーターとしての機能を有する塩基配列  (c) a base sequence comprising a part of the base sequence of (a) and having a function as a promoter substantially the same as the promoter comprising the DNA of (a)
〔11〕さらに、植物の木繊維形成組織に特異的発現に関与する転写因子を認識する 塩基配列を機能的に結合させた、〔10〕に記載のプロモーター DNA。  [11] The promoter DNA according to [10], further comprising a base sequence that recognizes a transcription factor involved in specific expression in a tree fiber-forming tissue of a plant.
〔12〕〔1〕〜〔3〕、〔10〕または〔11〕のいずれかに記載の DNAを有する組換えべクタ  [12] A recombinant vector comprising the DNA according to any one of [1] to [3], [10] or [11]
〔13〕〔12〕に記載のベクターを含むプラスミドを保持する微生物。 [13] A microorganism carrying a plasmid comprising the vector according to [12].
〔14〕〔12〕に記載のベクターが導入された形質転換植物細胞。 〔15〕〔14〕に記載の形質転換植物細胞から再分化した形質転換植物体。 [14] A transformed plant cell into which the vector according to [12] has been introduced. [15] A transformed plant regenerated from the transformed plant cell according to [14].
〔16〕 [15]に記載の形質転換植物体の子孫またはクローンである、形質転換植物体  [16] A transformed plant that is a descendant or clone of the transformed plant according to [15]
〔17〕〔15〕または〔16〕に記載の形質転換植物体の繁殖材料。 [17] A propagation material for the transformed plant according to [15] or [16].
発明の効果  The invention's effect
[0030] 本発明の DNAを利用することにより、植物の木繊維形成を人為的に制御し、特には その本質である細胞壁成分（セルロース ·へミセルロース ·リグニン）の量並びに質を 可変すること、さらに繊維形態 (木繊維細胞の伸長'細胞壁の厚さ）を自由に可変す ること、さらに木繊維特異的な遺伝子発現制御が可能となった。即ち、木質バイオマ スの量的な増大並びに質的な改良が見込まれ、これによつてパルプ生産と製紙の効 率ィ匕ゃ新規な特性を持った紙の製造が期待できる。カロえて、将来のエネルギー利用 や工業原料としての活用における用途の拡張も期待できる。  [0030] By using the DNA of the present invention, artificially controlling the formation of plant fiber, and in particular, changing the quantity and quality of cell wall components (cellulose, hemicellulose, lignin) that are the essence of the plant. Furthermore, the fiber morphology (elongation of wood fiber cells'cell wall thickness) can be freely changed, and gene expression control specific to wood fiber has become possible. In other words, it is expected that woody biomass will increase in quantity and quality will be improved, and as a result, pulp production and papermaking efficiency can be expected to produce paper with new characteristics. In addition, it can be expected to expand applications for future use of energy and utilization as industrial raw materials.
[0031] また、本発明の DNAは、植物の榭幹木部の検査用マーカーとして利用することがで きる。本発明の DNAを用いることで、植物の榭幹木部の生長状態、木繊維の形成状 態、ノルプ品質の検査、および有用な形質を有する植物の選抜ができる。  [0031] Furthermore, the DNA of the present invention can be used as a test marker for trunk stem parts of plants. By using the DNA of the present invention, it is possible to examine the growth state of trunk trunks of trees, the formation state of wood fibers, the quality of norp quality, and the selection of plants having useful traits.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0032] 本発明は、植物樹幹の木繊維細胞壁を形成する機能を有するタンパク質 (セル口 ース合成に関与するタンパク質、およびリグニン合成に関与するタンパク質)をコード する DNAとプロモーター DNAを提供する。  [0032] The present invention provides a DNA encoding a protein having a function of forming a tree fiber cell wall of a plant trunk (a protein involved in cell mouth synthesis and a protein involved in lignin synthesis) and a promoter DNA.
[0033] 本発明における植物樹幹の木繊維細胞壁を形成する機能を有するタンパク質をコ ードする DNAは、植物の木繊維細胞形成の制御に利用できると考えられる。一方、 該 DNAのプロモーター DNAは、木繊維組織あるいは木繊維形成時期特異的発現の 制御に利用できると考えられる。また、植物樹幹の木繊維細胞壁を形成する機能を 有するタンパク質をコードする DNAの発現 (レベル)情報は、植物体の生長状態や木 繊維形成状態、材質の量的'質的な事前予測に利用できると考えられる。従って、こ れらを用いることで、人為的な木繊維細胞形態形成の改変や任意の遺伝子'タンパ ク質の木繊維細胞特異的発現、植物体の生長状態や木繊維形成状態、材質の量的 •質的な事前予測に利用することができると考えられる。その結果、パルプ製造の効 率化、コストの削減、新規な特性を持った紙の製造が可能になる。 [0033] It is considered that DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk in the present invention can be used for control of plant fiber fiber cell formation. On the other hand, it is considered that the promoter DNA of the DNA can be used to control expression specific to wood fiber tissue or wood fiber formation time. In addition, the expression (level) information of DNA encoding a protein that functions to form the tree fiber cell wall of plant trunks is used for quantitative and qualitative prediction of plant growth, fiber formation, and materials. It is considered possible. Therefore, by using these, artificial modification of tree fiber cell morphogenesis, specific expression of protein's tree fiber cells, expression of plant growth state, state of fiber formation, amount of material • It can be used for qualitative advance prediction. As a result, the effectiveness of pulp production It will be possible to increase efficiency, reduce costs, and produce paper with new characteristics.
[0034] 植物において木繊維形成を制御することは、工業や農業の分野において、様々な 重要な意義を有する。例えば、ユーカリ属カマルドレンシス種の木繊維性質の改変 は、繊維長を長くすることによりパルプ等繊維原材料の繊維特性の向上などの点で 有意義である。またユーカリ属グロブルス種のセルロース ·へミセルロース含量を高め ることによるパルプ等の収量増大や、蒸解効率の向上などをもたらし、経済性や収益 性の点で有意義である。  [0034] Controlling wood fiber formation in plants has various important implications in the fields of industry and agriculture. For example, modification of the wood fiber properties of Eucalyptus genus Camaldrensis is significant in terms of improving the fiber properties of fiber raw materials such as pulp by increasing the fiber length. In addition, increasing the cellulose and hemicellulose content of Eucalyptus globulae species increases the yield of pulp and the like, and improves the cooking efficiency, which is significant in terms of economy and profitability.
[0035] 本発明の DNAの由来する植物としては、特に制限はなぐ例えば、穀類、野菜、果 榭等の有用農作物 (飼料作物を含む)、パルプ等の繊維原材料植物、観葉植物等の 鑑賞用植物等が挙げられる。該植物としては特に制限はなぐユーカリ、マツ、ァカシ ァ、ポプラ、スギ、ヒノキ、タケ、ィチイ、イネ、トウモロコシ、コムギ、ォォムギ、ライムギ 、ジャガイモ、タバコ、サトウダイコン、サトウキビ、ナタネ、ダイズ、ヒマヮリ、ヮタ、ォレ ンジ、ブドウ、モモ、ナシ、リンゴ、トマト、ハクサイ、キャベツ、ダイコン、ニンジン、カボ チヤ、キユウリ、メロン、パセリ、ラン、キク、ユリ、サフラン等を例示することができる。  [0035] The plant from which the DNA of the present invention is derived is not particularly limited, for example, useful crops (including forage crops) such as cereals, vegetables, fruit berries, fiber raw material plants such as pulp, ornamental plants, etc. Examples include plants. There are no particular restrictions on the plant, such as eucalyptus, pine, acacia, poplar, cedar, cypress, bamboo, yew, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower, Examples include ivy, orange, grape, peach, pear, apple, tomato, Chinese cabbage, cabbage, Japanese radish, carrot, cabbage, cucumber, melon, parsley, orchid, chrysanthemum, lily and saffron.
[0036] 本発明にお ヽて、植物樹幹の木繊維細胞壁を形成する機能を有するタンパク質を コードする DNAとしては、以下の（a)〜（e)のいずれかに記載の DNAが挙げられる。 [0036] In the present invention, examples of the DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree include the DNAs described in any of (a) to (e) below.
(a)配列番号： 35〜68のいずれかに記載のアミノ酸配列力 なるタンパク質をコード する DNA (a) DNA encoding a protein having the amino acid sequence ability described in any of SEQ ID NOs: 35 to 68
(b)配列番号： 1〜34のいずれかに記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 1-34
(c)配列番号： 35〜68のいずれかに記載のアミノ酸配列力 なるタンパク質と 50%以 上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with a protein having an amino acid sequence ability according to SEQ ID NO: 35-68
(d)配列番号： 1〜34のいずれかに記載の塩基配列力 なる DNAとストリンジェントな 条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence according to SEQ ID NO: 1-34
(e)配列番号： 35〜68のいずれかに記載のアミノ酸配列において 1または複数のァ ミノ酸が置換、欠失、付加、および/または挿入されたアミノ酸配列からなるタンパク 質をコードする DNA  (e) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 35-68
[0037] 本発明において、ストリンジェントなハイブリダィゼーシヨン条件としては、 O.lxSSC溶 液中にて、 60°C、ー晚放置の条件またはこれと同等のストリンジエンシーのノ、イブリダ ィゼーシヨン条件が挙げられる。このような条件において、配列番号： 1〜34のいずれ かに記載の塩基配列力もなる DNAとハイブリダィズする DNAが単離できる。 [0037] In the present invention, stringent hybridization conditions include conditions of 60 ° C, standing at room temperature in an O.lxSSC solution, or an equivalent stringency of no ibrida. An example is the condition. Under such conditions, DNA that hybridizes with DNA having the nucleotide sequence ability described in any one of SEQ ID NOs: 1 to 34 can be isolated.
[0038] 具体的には、植物より、 DNAを抽出し、遺伝子ライブラリーを構築し、同様の条件に よりスクリーニングを行うか、もしくは、抽出した DNAに対して、配列番号： 1〜34のい ずれかに記載の塩基配列より、任意の 20mer程度の配列をプライマーに用いて、劉ら によって確立された TAIL-PCR法により、連続する近傍配列を容易に取得できる。  [0038] Specifically, DNA is extracted from a plant, a gene library is constructed, and screening is performed under the same conditions, or the extracted DNA is represented by SEQ ID NOs: 1-34. From the base sequence described in any of the sequences, a contiguous neighboring sequence can be easily obtained by the TAIL-PCR method established by Liu et al. Using an arbitrary 20-mer sequence as a primer.
[0039] また、本発明は、配列番号： 35〜68のいずれかに記載のアミノ酸配列力 なるタン ノ ク質と 50%以上の相同性を有するタンパク質をコードする DNAも提供する。このよう な DNAは、当業者においては、一般的に公知の方法により単離することが可能であ る。例えば、ハイブリダィゼーシヨン技術（Southern, EM., J Mol Biol, 1975, 98, 503.) やポリメラーゼ連鎖反応（PCR)技術（Saiki, RK. et al., Science, 1985, 230, 1350.、 Sa iki, RK. et al., Science 1988, 239, 487.)を利用する方法が挙げられる。すなわち、配 列番号： 1〜34のいずれかに記載の塩基配列からなる DNAもしくはその一部をプロ ーブとして、また、配列番号： 1〜34のいずれかに記載の塩基配列からなる DNAに特 異的にハイブリダィズするオリゴヌクレオチドをプライマーとして、植物力 配列番号： 1〜34のいずれかに記載の塩基配列からなる DNAと高い相同性を有する DNAを単 離することは、当業者にとって通常行い得ることである。  [0039] The present invention also provides a DNA encoding a protein having 50% or more homology with a protein having the amino acid sequence ability described in any of SEQ ID NOs: 35 to 68. Such DNA can be isolated by those skilled in the art by generally known methods. For example, hybridization technology (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR) technology (Saiki, RK. Et al., Science, 1985, 230, 1350. Sa iki, RK. Et al., Science 1988, 239, 487.). That is, the DNA consisting of the base sequence described in any one of SEQ ID NOs: 1-34 or a part thereof as a probe, and the DNA consisting of the base sequence described in any one of SEQ ID NOs: 1-34 It is a common practice for those skilled in the art to isolate a DNA having a high homology with a DNA comprising the base sequence described in any one of SEQ ID NOS: 1 to 34 using an oligonucleotide that specifically hybridizes as a primer. Is to get.
[0040] このような DNAを単離するためには、好ましくはストリンジェントな条件下でハイブリ ダイゼーシヨン反応を行う。本発明にお 、てストリンジェントなハイブリダィゼーシヨン 条件とは、 6M尿素、 0.4% SDS、 0.5 X SSCの条件またはこれと同等のストリンジェン シ一のハイブリダィゼーシヨン条件を指す。よりストリンジエンシーの高い条件、例えば 、 6M尿素、 0.4% SDS、 0.1 X SSCの条件下では、より相同性の高い DNAを単離でき ると期待される。こうして単離された DNAは、アミノ酸レベルにおいて配列番号： 1〜3 4のいずれかに記載の塩基配列力もなる DNAからコードされるアミノ酸配列と高い相 同性を有すると考えられる。高い相同性とは、アミノ酸配列全体で少なくとも 50%以上 、好ましくは 70%以上、より好ましくは 80%以上、さらに好ましくは 90%以上、さらによ り好ましくは 95%以上、最も好ましくは 98%以上の配列の同一性を指す。  [0040] In order to isolate such DNA, a hybridization reaction is preferably performed under stringent conditions. In the present invention, stringent hybridization conditions refer to conditions of 6M urea, 0.4% SDS, 0.5 X SSC or equivalent stringency hybridization conditions. It is expected that DNA with higher homology can be isolated under conditions with higher stringency, for example, 6M urea, 0.4% SDS, and 0.1X SSC. The DNA thus isolated is considered to have high homology with the amino acid sequence encoded by the DNA having the nucleotide sequence described in any one of SEQ ID NOs: 1 to 34 at the amino acid level. High homology means at least 50% or more of the entire amino acid sequence, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more. Refers to the identity of the sequence.
[0041] アミノ酸配列や塩基配列の同一性は、カーリンおよびアルチユールによるアルゴリズ ム BLAST (Proc. Natl. Acad. Sci. USA, 1990, 87, 2264-2268.、 Karlin, S. & Altschul, SF" Proc. Natl. Acad. Sci. USA, 1993, 90, 5873.)を用いて決定できる。 BLASTのァ ルゴリズムに基づ 、た BLASTNや BLASTXと呼ばれるプログラムが開発されて!、る (A1 tschul, SF. et al.， J Mol Biol, 1990, 215, 403.) 0 BLASTNを用いて塩基配列を解析 する場合は、パラメータ一は、例えば score= 100、 wordlength= 12とする。また、 BLA STXを用いてアミノ酸配列を解析する場合は、パラメータ一は、例えば score = 50、 wo rdlength=3とする。 BLASTと Gapped BLASTプログラムを用いる場合は、各プログラム のデフォルトパラメーターを用いる。これらの解析方法の具体的な手法は公知である (http://www.ncbi.nlm.nih.gov/)。 [0041] The identity of the amino acid sequence and base sequence is determined by the algorithm by Carlin and Arthur. BLAST (Proc. Natl. Acad. Sci. USA, 1990, 87, 2264-2268., Karlin, S. & Altschul, SF "Proc. Natl. Acad. Sci. USA, 1993, 90, 5873.) Based on the BLAST algorithm, programs called BLASTN and BLASTX have been developed !, (A1 tschul, SF. Et al., J Mol Biol, 1990, 215, 403.) 0 BLASTN When analyzing the nucleotide sequence using the parameter 1, the parameter 1 is, for example, score = 100 and wordlength = 12. When analyzing the amino acid sequence using BLA STX, the parameter 1 is, for example, score = 50, wo rdlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used, and specific methods for these analysis methods are known (http: //www.ncbi.nlm.nih. gov /).
[0042] また、本発明の DNAには、配列番号： 35〜68のいずれかに記載のアミノ酸配列に おいて 1または複数のアミノ酸が置換、欠失、付加、および/または挿入されたァミノ 酸配列からなるタンパク質をコードする DNAが含まれる。  [0042] The DNA of the present invention includes an amino acid in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 35 to 68. DNA encoding a protein consisting of a sequence is included.
[0043] 上記の DNAを調製するために、当業者によく知られた方法としては、例えば、 DNA に对し、 site-directed mutagenesis法 (Kramer, W. & Fritz, HJ" Methods Enzymol, 1 987, 154, 350.)により変異を導入する方法が挙げられる。  [0043] To prepare the above DNA, methods well known to those skilled in the art include, for example, the site-directed mutagenesis method (Kramer, W. & Fritz, HJ "Methods Enzymol, 1 987 , 154, 350.).
[0044] タンパク質におけるアミノ酸の改変は、通常、全アミノ酸の 50アミノ酸以内であり、好 ましくは 30アミノ酸以内であり、さらに好ましくは 10アミノ酸以内であり、さらに好ましく は 3アミノ酸以内である。アミノ酸の改変は、例えば、変異や置換であれば「Transform er Site— directed Mutagenesis Kit」や「ExSite PCR— Based ¾ite— directed Mutagenesis KitJ (Clontech社製）を用いて行うことが可能であり、また、欠失であれば「Quantum le ap Nested Deletion KitJ (Clontech社製）などを用いて行うことが可能である。  [0044] Modification of amino acids in a protein is usually within 50 amino acids of all amino acids, preferably within 30 amino acids, more preferably within 10 amino acids, and even more preferably within 3 amino acids. The amino acid modification can be performed using, for example, “Transformer Site—directed Mutagenesis Kit” or “ExSite PCR—Based ¾ite—directed Mutagenesis KitJ (Clontech), for mutation or substitution, Deletion can be performed using “Quantum le ap Nested Deletion Kit J (Clontech)”.
[0045] また、塩基配列が変異していても、その変異がタンパク質中のアミノ酸の変異を伴 わない場合 (縮重変異）があり、このような縮重変異 DNAも本発明に含まれる。  [0045] Further, even if the base sequence is mutated, there is a case where the mutation is not accompanied by a mutation of an amino acid in the protein (degenerate mutation), and such degenerate mutated DNA is also included in the present invention.
[0046] 本発明の DNAは、本発明のタンパク質をコードし得るものであれば特に制限はなく 、ゲノム DNA、 cDNA、化学合成 DNAなどが含まれる。ゲノム DNAは、例えば、文献（R ogers and Bendich, Plant Mol. Biol, 1985, 5, 69.)記載の方法に従って調製したゲノ ム DNAを铸型として、本発明の DNAの塩基配列（例えば、配列番号： 1〜34のいず れかに記載の塩基配列）を基に作製したプライマーを用いて PCR (Saiki et al. Scienc e, 1988, 239, 487.)を行うことにより調製することが可能である。また、 cDNAであれば 、常法 (Maniatis et al. Molecular Cloning Cold Spring harbor Laboratry Press)により 植物から mRNAを調製し、逆転写反応を行い、上記と同様のプライマーを用いて PCR を行うことにより調製することが可能である。また、ゲノム DNAや cDNAは、常法により ゲノム DNAライブラリーまたは cDNAライブラリーを作製し、このライブラリーに対し、例 えば本発明の DNAの塩基配列（例えば、配列番号： 1〜34のいずれかに記載の塩 基配列）を基に合成したプローブを用いてスクリーニングすることによつても調製する ことが可能である。なお、得られた DNAの塩基配列は、例えば「シークェンサ一 Model 373」（ABI社製)を利用することにより容易に決定することが可能である。 [0046] The DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, cDNA, chemically synthesized DNA, and the like. Genomic DNA is, for example, a genomic DNA prepared according to the method described in the literature (Rogers and Bendich, Plant Mol. Biol, 1985, 5, 69.) as a saddle type, and the base sequence of the DNA of the present invention (for example, sequence No .: PCR (Saiki et al. Scienc) using a primer prepared on the basis of the nucleotide sequence described in any of 1 to 34) e, 1988, 239, 487.). In the case of cDNA, it is prepared by preparing mRNA from a plant by a conventional method (Maniatis et al. Molecular Cloning Cold Spring Harbor Laboratry Press), performing a reverse transcription reaction, and performing PCR using the same primers as described above. Is possible. For genomic DNA or cDNA, a genomic DNA library or cDNA library is prepared by a conventional method. For example, the base sequence of the DNA of the present invention (for example, any one of SEQ ID NOs: 1-34) is prepared against this library. It can also be prepared by screening using a probe synthesized based on the base sequence described in (1). The base sequence of the obtained DNA can be easily determined by using, for example, “Sequencer Model 373” (manufactured by ABI).
[0047] 本発明にお ヽて、植物樹幹の木繊維細胞壁を形成する機能を有するタンパク質を コードする DNAには、後述の「ユーカリ属カマルドレンシスの榭幹基部および榭幹中 央部において、師部側と比較して木部側で発現レベルが上昇している DNA」、「ユー カリ属カマルドレンシスの榭幹基部にぉ 、て、師部側と比較して木部側で発現レベル が減少し、榭幹中央部において、師部側と比較して木部側で発現レベルが上昇して いる DNA」、「ユーカリ属カマルドレンシスの榭幹中央部において、師部側と比較して 木部側で発現レベルが上昇し、榭幹基部において、師部側と木部側で発現レベル が同等な DNA」、「ユーカリ属カマルドレンシスおよびユーカリ属グロブルスの樹幹に おいて、師部側と比較して木部側で発現レベルが上昇している DNA」、「ユーカリ属 カマルドレンシスの樹幹にぉ 、て、師部側と比較して木部側で発現レベルが上昇し、 ユーカリ属グロブルスの樹幹にぉ 、て、師部側と比較して木部側で発現レベルが減 少している DNA」、「ユーカリ属カマルドレンシスの榭幹において、師部側と比較して 木部側で発現レベルが上昇し、ユーカリ属グロブルスの榭幹において、師部側と木 部側で発現レベルが同等な DNA」が含まれる。  [0047] In the present invention, the DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk is described below in the "root base of Eucalyptus genus Camaldrensis" and the center of the trunk. DNA whose expression level is increased on the xylem side compared to the phloem side ”,“ The expression level on the xylem side compared to the phloem side, ” In the central trunk, the expression level is higher on the xylem side compared to the phloem side, "in the central trunk of Eucalyptus genus Camaldrensis compared to the phloem side. The expression level rises on the xylem side, and in the trunk trunk base, the DNA expression level is the same on the phloem side and the xylem side '', `` the phloem in the stem of Eucalyptus genus Camaldrensis and Eucalyptus globules DNA whose expression level is higher on the xylem side than on the other side ”,“ The expression level rises on the xylem side compared to the phloem side, and the tree level on the eucalyptus globulus trunk, compared to the phloem side. In the trunk of Eucalyptus genus Camaldrensis, the expression level increased on the xylem compared to the phloem side, and in the trunk of the Eucalyptus globules “DNA with the same expression level on the part side and xylem side” is included.
[0048] 榭幹において、師部と木部では、木部側でのみ木繊維形成が行われることが知ら れている。よって、「師部側と比較して木部側で発現レベルが上昇または減少してい る DNA」は、「木繊維形成部位で発現レベルが上昇または減少して ヽる DNA」と表現 することもできる。また、榭幹基部の木部と榭幹中央部の木部では、榭幹基部の木部 と比較して榭幹中央部の木部の方が、木繊維形成 (性質)のうち木繊維長は長ぐ直 径は太ぐ細胞壁は厚ぐフィブリル傾角は緩ぐ木繊維率と比重は高くなる（成熟材 である）ことが知られている。また、榭幹中央部の木部と比較して榭幹基部の木部の 方力 木繊維形成のうち木繊維長は短ぐ直径は細ぐ細胞壁は薄ぐフィブリル傾角 は急で、木繊維率と比重は低くなる（未熟材である）ことが知られている。よって、「ュ 一カリの榭幹基部にぉ 、て、師部側と比較して木部側で発現レベルが上昇または減 少している DNA」は、「未熟材形成部位で発現レベルが上昇または減少している DNA 」と表現することもできる。また、「ユーカリの榭幹中央部において、師部側と比較して 木部側で発現レベルが上昇または減少している DNA」は、「成熟材形成部位で発現 レベルが上昇または減少して ヽる DNA」と表現することもできる。 [0048] In the trunk, it is known that the phloem and the xylem form the wood fiber only on the xylem side. Therefore, “DNA whose expression level increases or decreases on the xylem side compared to the phloem side” can also be expressed as “DNA whose expression level increases or decreases on the wood fiber formation site”. it can. In addition, in the timber of the trunk base and the timber of the trunk trunk, the timber of the trunk trunk is more of the fiber length (characteristic) than the timber of the trunk trunk. Is long It is known that thicker cell walls are thicker and fibril inclination is looser, and the wood fiber ratio and specific gravity are higher (mature material). Also, compared with the xylem in the middle of the trunk, the xylem of the trunk of the trunk base Among the wood fiber formation, the wood fiber length is shorter, the diameter is thinner, the cell wall is thinner, the fibril inclination is steep, and the wood fiber rate It is known that the specific gravity is low (it is an immature material). Therefore, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side on the trunk base of a single pot” is expressed or increased in the immature wood formation site. It can also be expressed as “decreasing DNA”. In addition, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side in the central part of the eucalyptus trunk” is expressed in “ It can also be expressed as “DNA”.
[0049] さらに、カマルドレンシス榭幹の木部とグロブルス榭幹の木部では、カマルドレンシ ス榭幹の木部よりグロブルス榭幹の木部の方力 木繊維形成 (パルプ品質）のうち木 繊維長は長ぐ直径は太ぐ細胞壁は厚ぐフィブリル傾角は緩ぐ木繊維率と比重は 高くなることが知られている。また、グロブルス榭幹の木部よりカマルドレンシス榭幹の 木部の方が、木繊維長は短ぐ直径は細ぐ細胞壁は薄ぐフィブリル傾角は急で、 木繊維率と比重は低くなることが知られている。よって、「ユーカリ属カマルドレンシス の榭幹にぉ 、て、師部側と比較して木部側で発現レベルが上昇または減少して 、る DNA」は、「木繊維長は短ぐ直径は細ぐ細胞壁は薄ぐフィブリル傾角は急で、木 繊維率と比重は低 ヽ、と!ヽぅ低品質なパルプ特性を有する木繊維形成部位で発現レ ベルが上昇または減少している DNA」と表現することもできる。また、「ユーカリ属グロ プルスの樹幹にぉ 、て、師部側と比較して木部側で発現レベルが上昇または減少し ている DNA」は、「木繊維長は長ぐ直径は太ぐ細胞壁は厚ぐフィブリル傾角は緩く 、木繊維率と比重は高い、という高品質なパルプ特性を有する木繊維を形成する部 位で発現レベルが上昇または減少して ヽる DNA」と表現することもできる。  [0049] In addition, in the xylem of the Camaldrensis trunk and the grouse of the Globulus trunk, the direction of the xylem of the Grobles trunk from the xylem of the Camaldrensis trunk is the fiber of the wood fiber formation (pulp quality) It is known that the length and diameter are thick and the cell wall is thick and the fibril inclination is loose and the wood fiber ratio and specific gravity are high. Also, the xylem of the Camaldrensis trunk is shorter than the globules trunk, but the diameter of the fiber is shorter, the cell wall is thinner, the fibril inclination is steeper, and the wood fiber ratio and specific gravity are lower. It has been known. Therefore, “DNA that has an increased or decreased expression level on the xylem side compared to the phloem side, compared to the trunk of the Eucalyptus genus Camaldrensis” `` The thin cell walls are thin, the fibril inclination is steep, the wood fiber rate and specific gravity are low, and the expression level is increased or decreased at the wood fiber formation site with low quality pulp characteristics. '' It can also be expressed. In addition, “DNA with an increased or decreased expression level on the xylem side compared to the phloem side on the trunk of Eucalyptus globulus” means that “wood fiber length is longer and diameter is thicker than the cell wall. It can be expressed as `` DNA whose expression level is increased or decreased in the part that forms wood fiber with high quality pulp characteristics that thick fibril inclination is gentle, wood fiber rate and specific gravity are high '' .
[0050] 本発明にお 、て、師部側とは***組織である維管束形成層より外側を意味し、好 ましくは師部側の榭皮形成部位を意味する。ここで、榭皮形成部位とは師細胞、師管 、柔細胞、じん皮細胞等へ分ィ匕中またはその分ィ匕能力を有する部位である。また、本 発明において、木部側とは維管束形成層より内側を意味し、好ましくは木部側の木 繊維形成部位を意味する。ここで、木繊維形成部位とは木繊維細胞、仮導管、導管 、柔細胞等へ分ィ匕中またはその分ィ匕能力を有する部位である。 [0050] In the present invention, the phloem side means the outside of the vascularization layer, which is a meristem, and preferably means the scab formation site on the phloem side. Here, the scab formation site is a site that is in the middle of or is capable of splitting into phloem cells, phloem tubules, parenchyma cells, and pelvic cells. Further, in the present invention, the xylem side means the inner side of the vascular bundle forming layer, preferably the xylem side wood fiber forming site. Here, the wood fiber formation site is a wood fiber cell, temporary conduit, conduit In other words, it is a part that is in the middle of separating into parenchymal cells or the like or has the ability to separate the cells.
[0051] 本発明において、榭幹基部とは、一般的にいわれる胸高部未満を指し、例えば、ュ 一カリの 5年生の植物体の場合、地上高約 lm未満を指す。また、榭幹中央部とは、 一般的に榭高の中間部付近を指し、例えば、榭高 10m以上のユーカリの 5年生の植 物体の場合、地上高約 5m付近を指す。 [0051] In the present invention, the trunk base means a generally called breast height, for example, in the case of a 5- year-old plant of Eucalyptus, it indicates a ground height of less than about lm. In addition, the central part of the trunk generally refers to the middle part of the culm height, for example, in the case of a eucalyptus fifth-year plant that has a culm height of 10 m or more, it refers to a height of about 5 m above the ground.
[0052] 本発明の DNAまたはその部分 DNAは、植物の榭幹木部 (好ましくは榭幹基部また は榭幹中央部の木部）の生長状態の検査用マーカーとして利用できる。すなわち、 上記 DNAまたはその部分 DNAを少なくとも 1つ利用することで、植物の榭幹木部 (好 ましくは榭幹基部または榭幹中央部の木部)の生長状態を検査することができる。  [0052] The DNA of the present invention or a partial DNA thereof can be used as a marker for examining the growth state of a trunk trunk (preferably a trunk of a trunk trunk or a middle section of a trunk) of a plant. That is, by using at least one of the above-mentioned DNAs or partial DNAs thereof, the growth state of the trunk trunk of the plant (preferably the trunk of the trunk trunk or the central part of the trunk) can be examined.
[0053] より具体的には、以下の（1)〜 (4)の場合に、植物の榭幹木部が、木繊維形成が盛 んな木部であると判定される。さらに、（2)の場合に、植物の榭幹木部が、未熟材が 形成されて ヽる木部、または将来的に未熟材が形成される木部であると判定される。 ここで、未熟材とは、未熟な形成層組織から分化'形成される木繊維を意味し、同様 な条件で採取された榭幹中央部の木部と比較して、木繊維長は短ぐ直径は細ぐ 細胞壁は薄ぐフィブリル傾角は急で、木繊維率と比重は低いという特徴を有する。ま た、（3)または (4)の場合に、植物の榭幹木部が、成熟材が形成されている木部、ま たは将来的に成熟材が形成される木部であると判定される。ここで、成熟材とは、成 熟した形成層組織から分化して形成される木繊維を意味し、同様な条件で採取され た榭幹基部の木部と比較して、木繊維長は長ぐ直径は太ぐ細胞壁は厚ぐフイブリ ル傾角は緩ぐ木繊維率と比重は高 ヽと ヽぅ特徴を有する。  More specifically, in the following cases (1) to (4), it is determined that the trunk trunk portion of the plant is a xylem portion in which wood fiber formation is active. Furthermore, in the case of (2), it is determined that the trunk stem portion of the plant is a xylem portion in which immature wood is formed or a xylem in which immature material is formed in the future. Here, the immature wood means a wood fiber that is differentiated and formed from an immature formed layer structure, and the length of the wood fiber is shorter than that of the xylem of the center of the trunk collected under the same conditions. The diameter is thin. The cell wall is thin. The fibril inclination is steep, and the wood fiber ratio and specific gravity are low. In the case of (3) or (4), it is determined that the trunk trunk of the plant is a xylem where mature wood is formed, or a xylem where mature wood is formed in the future. Is done. Here, matured wood means wood fibers that are formed by differentiation from a mature formation layer structure, and the length of the wood fibers is longer than that of the trunk of the trunk base collected under the same conditions. The cell diameter is thick, the cell wall is thick, the fiber inclination is loose, and the wood fiber ratio and specific gravity are high and low.
[0054] 本発明の検査に用いられる植物は、好ましくはユーカリ、より好ましくはユーカリ属カ マルドレンシス種またはユーカリ属グロブルス種である。  [0054] The plant used in the test of the present invention is preferably Eucalyptus, more preferably Eucalyptus Camaldlensis or Eucalyptus Globulus.
[0055] また、本発明の検査方法を利用することで材質の量的 ·質的な事前予測が可能とな る。また、植林事業において、生長状態をもとに適切な施業 (除草'間伐等)を適切な 時期に施すことができる。例えば、本発明における榭幹基部の師部と木部での発現 ノターンと本発明の検査方法で得られた発現パターンに相関があれば (好ましくはピ ァソンの相関係数 0.5以上、より好ましくは 0.7以上、以下同じ）、被検榭幹木部に、質 的には未熟な繊維 (未熟材:短繊維、直径は細ぐ細胞壁は薄ぐフィブリル傾角は 急で、低木繊維率と低比重）が形成されている、もしくは将来的に形成される状態で あり、木繊維量 (容積重)も少な 、もしくは将来的に少なくなる（平均的な容積重 450 〜500kg/m3以下）という予測ができる。一方、本発明における榭幹中央部の師部と木 部での発現パターンと本発明の検査方法で得られた発現パターンに相関があれば、 被検榭幹木部に、質的には成熟した繊維 (成熟材:長繊維、直径は太ぐ細胞壁は 厚ぐフィブリル傾角は緩ぐ高木繊維率と高比重)が形成されている、もしくは将来 的に形成される状態であり、木繊維量 (容積重)も多いもしくは将来的に多くなる（平 均的な容積重 450〜500kg/m3以上）という予測ができる。さらに、上記のような材質予 測により、植物の生長状態の善し悪しが明ら力となるため、悪いと判断されれば、貧 栄養地では施肥を行ったり、雑草が茂っていれば除草作業を早期に行ったり、植栽 密度が高ければ間伐を行ったり、といった施業を迅速かつ適切に行うことが可能にな る。現在、前述のような材質予測や施業方針の決定は、多分に現場の経験や勘、も しくは事後の化学分析等に頼っており、本願で示すような事前に判別できる科学的 指標は無い。 [0055] Further, by using the inspection method of the present invention, quantitative and qualitative advance prediction of a material becomes possible. In afforestation projects, appropriate operations (weeding, thinning, etc.) can be carried out at appropriate times based on the growth conditions. For example, if there is a correlation between the expression pattern in the phloem and xylem of the trunk base in the present invention and the expression pattern obtained by the test method of the present invention (preferably the correlation coefficient of Pearson is 0.5 or more, more preferably 0.7 or more, the same shall apply hereinafter), qualitatively immature fibers (unmature wood: short fibers, thin diameter, thin cell walls, thin fibril inclination angle) Abruptly, shrub fiber ratio and low specific gravity) are formed, or in the future, and the amount of wood fiber (volume weight) is low or will decrease in the future (average volume weight 450 ~500kg / m 3 or less) that it is predictable. On the other hand, if there is a correlation between the expression pattern in the phloem and xylem of the center of the trunk in the present invention and the expression pattern obtained by the test method of the present invention, the maturity qualitatively in the subject trunk Fibers (mature material: long fibers, thicker cell walls are thicker, fibril inclination is thicker and higher wood fiber ratio and high specific gravity) are formed, or are formed in the future, and the amount of wood fibers ( It can be predicted that there will be a large (increase in specific gravity) or increase in the future (an average specific gravity of 450 to 500 kg / m 3 or more). Furthermore, the material quality prediction as described above will clearly reveal the quality of the plant's growth, so if it is judged to be bad, fertilizer is applied in an oligotrophic area or weeding work is carried out if weeds are thick. It is possible to conduct operations quickly and appropriately, such as early implementation or thinning if the planting density is high. Currently, material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance as shown in this application. .
(1)植物の榭幹 (好ましくは榭幹基部または榭幹中央部）の師部側および木部側に おいて、「ユーカリ属カマルドレンシスの榭幹基部および榭幹中央部において、師部 側と比較して木部側で発現レベルが上昇している DNA」のうち少なくとも 1つ (好ましく は複数、より好ましくは全て。以下同じ)の DNAの発現レベルを検出し、師部側および 木部側における該 DNAの発現レベルを比較した結果、植物の榭幹における該 DNA の発現レベル力 師部側と比較して木部側で上昇して 、る場合 (1) On the phloem side and the xylem side of the trunk of the plant (preferably the trunk trunk or the middle of the trunk), “the phloem in the trunk trunk and the middle trunk of the Eucalyptus genus Camaldrensis” The expression level of at least one (preferably a plurality, more preferably all, the same below) of DNA whose expression level is increased on the xylem side relative to the xylem side is detected. As a result of comparing the expression level of the DNA on the xylem side, the expression level of the DNA on the trunk of the plant is higher on the xylem side than on the phloem side
本発明にお 、て、「ユーカリ属カマルドレンシスの榭幹基部および榭幹中央部にお いて、師部側と比較して木部側で発現レベルが上昇している DNA」としては、以下の (a)〜（e)の!、ずれかに記載の DNAが挙げられる。  In the present invention, “DNA having an increased expression level on the xylem side compared to the phloem side in the trunk base and middle trunk of Eucalyptus genus Camaldrensis” Of (a) to (e), or any of the DNAs described in any of them.
(a)配列番号： 35〜40、 42、 43, 45〜48、 50、 51、 53〜59、 61、 62、 64, 66また は 67のいずれかに記載のアミノ酸配列からなるタンパク質をコードする DNA  (a) SEQ ID NO: 35 to 40, 42, 43, 45 to 48, 50, 51, 53 to 59, 61, 62, 64, 66 or 67 DNA
(b)配列番号： 1〜6、 8、 9、 11〜14、 16、 17、 19〜25、 27、 28、 30、 32または 33 の！、ずれかに記載の塩基配列を含む DNA  (b) SEQ ID NO: 1-6, 8, 9, 11-14, 16, 17, 19-25, 27, 28, 30, 32 or 33! DNA containing the nucleotide sequence described in any of
(c)配列番号： 35〜40、 42、 43, 45〜48、 50、 51、 53〜59、 61、 62、 64, 66また は 67のいずれかに記載のアミノ酸配列からなるタンパク質と 50%以上の相同性を有 するタンパク質をコードする DNA (c) SEQ ID NO: 35-40, 42, 43, 45-48, 50, 51, 53-59, 61, 62, 64, 66 Is a DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of any one of 67
(d)配列番号： 1〜6、 8、 9、 11〜14、 16、 17、 19〜25、 27、 28、 30、 32または 33 のいずれかに記載の塩基配列力 なる DNAとストリンジヱントな条件下でノヽイブリダィ ズする DNA  (d) SEQ ID NOs: 1 to 6, 8, 9, 11 to 14, 16, 17, 19 to 25, 27, 28, 30, 32, or 33 DNA having stringency and stringent conditions DNA noisy underneath
(e)配列番号： 35〜40、 42、 43, 45〜48、 50、 51、 53〜59、 61、 62、 64, 66また は 67のいずれかに記載のアミノ酸配列において 1または複数のアミノ酸が置換、欠 失、付加、および Zまたは挿入されたアミノ酸配列からなるタンパク質をコードする DN A  (e) SEQ ID NO: 35-40, 42, 43, 45-48, 50, 51, 53-59, 61, 62, 64, 66 or 67 DN A that encodes a protein consisting of a substitution, deletion, addition, and Z or inserted amino acid sequence
「配列番号： 1〜6、 8、 9、 11〜14、 16、 17、 19〜25、 27, 28, 30、 32または 33 のいずれかに記載の塩基配列からなる DNA」としては、配列番号： 1〜6、 9、 11〜14 、 16、 17、 19、 20、 22、 25、 27また ίま 30の!ヽずれ力に記載の塩基酉己歹 IJ力らなる DN Α力好ましく、酉己歹 IJ番号： 1、 3〜6、 9、 11〜14、 16、 17、 19、 22、 25また ίま 30の!ヽ ずれかに記載の塩基配列力 なる DNAがより好ましいが、これらに限定されるもので はない。  “SEQ ID NO: 1 to 6, 8, 9, 11 to 14, 16, 17, 19 to 25, 27, 28, 30, 32 or 33” as the DNA : 1 to 6, 9, 11 to 14, 16, 17, 19, 20, 22, 25, 27 or 30! Self IJ numbers: 1, 3-6, 9, 11-14, 16, 17, 19, 22, 25 or ί or 30! It is not limited.
(2)植物の榭幹 (好ましくは榭幹基部）の師部側および木部側において、「ユーカリ属 カマルドレンシスの榭幹基部にお 、て、師部側と比較して木部側で発現レベルが減 少し、榭幹中央部において、師部側と比較して木部側で発現レベルが上昇している DNA」のうち少なくとも 1つの DNAの発現レベルを検出し、師部側および木部側にお ける該 DNAの発現レベルを比較した結果、植物の榭幹における該 DNAの発現レべ ルが、師部側と比較して木部側で減少して 、る場合 (2) At the phloem side and the xylem side of the plant trunk (preferably the trunk trunk base), “In the trunk base of the Eucalyptus genus Camaldrensis, compared to the phloem side, The expression level decreases, and the expression level of at least one of the DNAs whose expression level is increased on the xylem side compared to the phloem side in the middle of the trunk is detected. When the expression level of the DNA on the xylem side is compared, the expression level of the DNA in the trunk of the plant is decreased on the xylem side compared to the phloem side.
本発明において、「ユーカリ属カマルドレンシスの榭幹基部において、師部側と比 較して木部側で発現レベルが減少し、榭幹中央部において、師部側と比較して木部 側で発現レベルが上昇して 、る DNA」としては、以下の（a)〜（e)の 、ずれかに記載 の DNAが挙げられる。  In the present invention, the expression level is reduced on the xylem side in the trunk base of Eucalyptus genus Camaldrensis, compared to the phloem side, and in the middle part of the trunk, the xylem side is compared with the phloem side. Examples of the DNA whose expression level is increased in (1) include the DNAs described in any one of the following (a) to (e).
(a)配列番号: 49に記載のアミノ酸配列力 なるタンパク質をコードする DNA  (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 49
(b)配列番号： 15に記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 15
(c)配列番号： 49に記載のアミノ酸配列力もなるタンパク質と 50%以上の相同性を有 するタンパク質をコードする DNA (c) 50% or more homology with the protein having the amino acid sequence ability described in SEQ ID NO: 49 DNA encoding the protein
(d)配列番号： 15に記載の塩基配列力 なる DNAとストリンジェントな条件下でノ、イブ リダィズする DNA  (d) DNA that has the nucleotide sequence of SEQ ID NO: 15 and DNA that can be hybridized under stringent conditions
(e)配列番号： 49に記載のアミノ酸配列にお 、て 1または複数のアミノ酸が置換、欠 失、付加、および Zまたは挿入されたアミノ酸配列からなるタンパク質をコードする DN A  (e) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence set forth in SEQ ID NO: 49
[0058] (3)植物の榭幹 (好ましくは榭幹中部）の師部側および木部側において、上記「ユー カリ属カマルドレンシスの榭幹基部にぉ 、て、師部側と比較して木部側で発現レベル が減少し、榭幹中央部において、師部側と比較して木部側で発現レベルが上昇して V、る DNA」のうち少なくとも 1つの DNAの発現レベルを検出し、師部側および木部側 における該 DNAの発現レベルを比較した結果、植物の榭幹における該 DNAの発現 レベル力 師部側と比較して木部側で上昇して 、る場合  [0058] (3) On the phloem side and the xylem side of the trunk of the plant (preferably in the middle of the trunk), compared to the phloem side of the eucalyptus genus Camaldrensis, The expression level decreases on the xylem side, and the expression level rises on the xylem side compared to the phloem side in the middle of the trunk, and the expression level of at least one DNA of `` V, ru DNA '' is detected When the expression level of the DNA on the phloem side and the xylem side is compared, the expression level of the DNA in the trunk of the plant is higher on the xylem side than on the phloem side.
[0059] (4)植物の榭幹 (好ましくは榭幹中央部）の師部側および木部側において、「ユーカリ 属カマルドレンシスの榭幹中央部にお 、て、師部側と比較して木部側で発現レベル が上昇し、榭幹基部において、師部側と木部側で発現レベルが同等な DNA」のうち 少なくとも 1つの DNAの発現レベルを検出し、師部側および木部側における該 DNA の発現レベルを比較した結果、植物の榭幹における該 DNAの発現レベル力 師部側 と比較して木部側で上昇して ヽる場合  [0059] (4) On the phloem side and the xylem side of the trunk of the plant (preferably in the middle of the trunk), compared with the phloem side in the middle of the trunk of Eucalyptus genus Camaldrensis. The expression level rises on the xylem side, and at the trunk base, the expression level of at least one of the DNAs with the same expression level on the phloem side and the xylem side is detected. As a result of comparing the expression level of the DNA on the side, the expression level of the DNA on the trunk of the plant
本発明において、「ユーカリ属カマルドレンシスの榭幹中央部において、師部側と 比較して木部側で発現レベルが上昇し、榭幹基部において、師部側と木部側で発現 レベルが同等な DNA」としては、以下の（a)〜（e)の!、ずれかに記載の DNAが挙げら れる。  In the present invention, the expression level is increased on the xylem side compared to the phloem side in the central trunk of Eucalyptus genus Camaldrensis, and the expression level is increased on the phloem side and xylem side in the trunk base. Examples of “equivalent DNA” include the DNAs described in the following (a) to (e)!
(a)配列番号： 41、 44、 52、 60、 63または 68のいずれかに記載のアミノ酸配列から なるタンパク質をコードする DNA  (a) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 41, 44, 52, 60, 63 or 68
(b)配列番号： 7、 10、 18、 26、 29または 34のいずれかに記載の塩基配列を含む D NA  (b) SEQ ID NO: 7, DNA comprising the nucleotide sequence set forth in any one of 18, 18, 26, 29 or 34
(c)配列番号： 41、 44、 52、 60、 63または 68のいずれかに記載のアミノ酸配列から なるタンパク質と 50%以上の相同性を有するタンパク質をコードする DNA (d)配列番号： 7、 10、 18、 26、 29または 34のいずれかに記載の塩基配列からなる DNAとストリンジェントな条件下でハイブリダィズする DNA (c) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence set forth in any of SEQ ID NOs: 41, 44, 52, 60, 63 or 68 (d) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 7, 10, 18, 26, 29, or 34
(e)酉己歹 IJ番号： 41、 44、 52、 60、 63また ίま 68の!ヽずれ力に記載のアミノ酸酉己歹 IJにお いて 1または複数のアミノ酸が置換、欠失、付加、および Ζまたは挿入されたアミノ酸 配列からなるタンパク質をコードする DNA  (e) Amino acids listed in the IJ numbers: 41, 44, 52, 60, 63 or ί or 68! One or more amino acids are substituted, deleted or added in IJ , And DNA encoding a protein consisting of Ζ or inserted amino acid sequence
「配列番号： 7、 10、 18、 26、 29または 34のいずれかに記載の塩基配列力 なる D ΝΑ」としては、配列番号： 7、 10、 18、 26または 29のいずれかに記載の塩基配列か らなる DNAが好ましぐ配列番号： 18、 26または 29のいずれかに記載の塩基配列か らなる DNAがより好まし!/、が、これらに限定されるものではな！/、。  “The base sequence ability D 記載 described in any of SEQ ID NOs: 7, 10, 18, 26, 29, or 34” includes the bases described in any of SEQ ID NOs: 7, 10, 18, 26, or 29. DNA comprising the sequence is preferred DNA comprising the base sequence described in SEQ ID NO: 18, 26 or 29 is more preferred! /, But is not limited to these! /.
さらに、本発明においては、異なる 2つの榭幹木部の生長状態 (成熟度)を検査す る方法を提供する。すなわち、異なる 2つの榭幹木部うち、どちらが成熟しているのか 、または未熟であるのかを検査する方法を提供する。本発明において、「異なる 2つ の榭幹木部」には、異なる 2つの植物における榭幹木部、および、 1つの植物におけ る異なる 2つの榭幹木部が含まれる。上記方法は、複数の榭幹木部から、成熟した、 または未熟な榭幹木部を選別することにも使用できる。  Furthermore, the present invention provides a method for examining the growth state (maturity) of two different trunks. In other words, it provides a method for examining which of two different trunks are mature or immature. In the present invention, “two different trunk trunks” include two trunk trunks in two different plants and two different trunk trunks in one plant. The above method can also be used to select mature or immature trunk trunks from a plurality of trunk trunks.
より具体的には、以下の（5)または（6)の場合に、異なる 2つの榭幹木部のうち、片 方の榭幹木部 (以下、榭幹木部 Αと称す)と比較して他方の榭幹木部 (以下、榭幹木 部 Bと称す）は、成熟している木部であり、榭幹木部 Bと比較して榭幹木部 Aは、未熟 な木部であると判定される。従って、以下の（5)または（6)は、例えば育種現場にお いて、エリート木やプラスツリーと呼ばれる優良系統を選抜するときの指標になる。現 在、エリート木等の選抜は、榭高ゃ直径などの外見上の数値や伐採後の材質分析 値を指標に行われており、上記指標で選抜する技術 (概念)は無い。  More specifically, in the case of (5) or (6) below, it is compared with one of the two different trunk trunks (hereinafter referred to as the trunk trunk trunk). The other trunk tree section (hereinafter referred to as trunk trunk section B) is a mature section. Compared with trunk trunk section B, trunk trunk section A is an immature section. It is determined that there is. Therefore, the following (5) or (6) is an index for selecting an excellent line called an elite tree or a plus tree, for example, at a breeding site. At present, selection of elite trees, etc. is performed using the numerical values such as the diameter of the spiders and diameters and the material analysis values after harvesting as indicators, and there is no technique (concept) to select with the above indicators.
(5)異なる 2つの榭幹木部において、「ユーカリの榭幹中央部の木部側と比較して榭 幹基部の木部側で発現レベルが上昇している DNA」のうち少なくとも 1つの DNAの発 現レベルを検出し、異なる 2つの榭幹木部における該 DNAの発現レベルを比較した 結果、該 DNAの発現レベル力 榭幹木部 Bと比較して榭幹木部 Aで上昇している場 本発明において、「ユーカリの榭幹中央部の木部側と比較して榭幹基部の木部側 で発現レベルが上昇して 、る DNA」としては、以下の（a)〜（e)の!、ずれかに記載の DNAが挙げられる。 (5) At least one DNA in two different trunk trunks, "DNA whose expression level is increased on the xylem side of the cocoon trunk compared to the xylem side of the eucalyptus trunk center" As a result of detecting the expression level of the DNA and comparing the expression level of the DNA in two different trunk trunks, the expression level of the DNA increased in the trunk trunk A compared to the trunk trunk B. In the present invention, “the xylem side of the trunk base compared to the xylem side of the eucalyptus trunk center” Examples of the DNA whose expression level is increased in (a) to (e) below include DNA described in any of!
(a)配列番号： 67または 68に記載のアミノ酸配列力もなるタンパク質をコードする DN A  (a) DN A encoding a protein having the amino acid sequence ability described in SEQ ID NO: 67 or 68
(b)配列番号： 33または 34に記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 33 or 34
(c)配列番号: 67または 68に記載のアミノ酸配列力もなるタンパク質と 50%以上の相 同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in SEQ ID NO: 67 or 68
(d)配列番号： 33または 34に記載の塩基配列からなる DNAとストリンジ ントな条件 下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 33 or 34
(e)配列番号： 67または 68に記載のアミノ酸配列において 1または複数のアミノ酸が 置換、欠失、付加、および Zまたは挿入されたアミノ酸配列からなるタンパク質をコー ドする DNA  (e) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence set forth in SEQ ID NO: 67 or 68
「配列番号： 33または 34に記載の塩基配列力 なる DNA」としては、配列番号： 33 に記載の塩基配列からなる DNAが好まし 、が、これらに限定されるものではな!/、。 (6)異なる 2つの榭幹木部において、「ユーカリの榭幹中央部の木部側と比較して榭 幹基部の木部側で発現レベルが減少している DNA」のうち少なくとも 1つの DNAの発 現レベルを検出し、異なる 2つの榭幹木部における該 DNAの発現レベルを比較した 結果、該 DNAの発現レベル力 榭幹木部 Bと比較して榭幹木部 Aで減少している場 本発明において、「ユーカリの榭幹中央部の木部側と比較して榭幹基部の木部側 で発現レベルが減少して 、る DNA」としては、以下の（a)〜（e)の!、ずれかに記載の DNAが挙げられる。  The “DNA having the nucleotide sequence described in SEQ ID NO: 33 or 34” is preferably a DNA consisting of the nucleotide sequence described in SEQ ID NO: 33, but is not limited to these! /. (6) At least one DNA in two different trunk trunks, "DNA whose expression level is reduced on the xylem side of the trunk base compared to the xylem side of the eucalyptus trunk center" As a result of comparing the expression level of the DNA in two different trunk trunks, the expression level of the DNA decreased in the trunk trunk A compared to the trunk trunk B. In the present invention, “the DNA whose expression level is reduced on the xylem side of the trunk base compared to the xylem side of the eucalyptus trunk center” is as follows: (a) to (e )! Or any of the DNAs listed elsewhere.
(a)配列番号： 35〜40、 42、 48、 50、 51、 53〜56、 58、 59、 62または 64のいずれ かに記載のアミノ酸配列力 なるタンパク質をコードする DNA  (a) DNA encoding the protein having the amino acid sequence ability according to any one of SEQ ID NOs: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62 or 64
(b)酉己歹 IJ番号： 1〜6、 8、 14、 16、 17、 19〜22、 24、 25、 28また ίま 30の!ヽずれ力に 記載の塩基配列を含む DNA  (b) IJ number: 1-6, 8, 14, 16, 17, 19-22, 24, 25, 28 or ί or 30!
(c)配列番号： 35〜40、 42、 48、 50、 51、 53〜56、 58、 59、 62または 64のいずれ かに記載のアミノ酸配列力もなるタンパク質と 50%以上の相同性を有するタンパク質 をコードする DNA (c) SEQ ID NO: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62 or 64 A protein having 50% or more homology with the protein having amino acid sequence ability DNA encoding
(d)酉己歹 IJ番号： 1〜6、 8、 14、 16、 17、 19〜22、 24、 25、 28また ίま 30の!ヽずれ力に 記載の塩基配列力もなる DNAとストリンジェントな条件下でハイブリダィズする DNA (d) IJ numbers: 1 to 6, 8, 14, 16, 17, 19 to 22, 24, 25, 28 or ί or 30! DNA that hybridizes under mild conditions
(e)配列番号： 35〜40、 42、 48、 50、 51、 53〜56、 58、 59、 62または 64のいずれ かに記載のアミノ酸配列において 1または複数のアミノ酸が置換、欠失、付加、およ び Zまたは挿入されたアミノ酸配列力もなるタンパク質をコードする DNA (e) SEQ ID NO: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62 or 64, one or more amino acids are substituted, deleted or added , And DNA encoding a protein that also has Z or inserted amino acid sequence ability
「酉己歹 IJ番号： 1〜6、 8、 14、 16、 17、 19〜22、 24、 25、 28また ίま 30の! /、ずれ力に 記載の塩基配列からなる DNA」としては、配列番号： 1〜6、 14、 16、 17、 19、 20、 2 2、 25または 30のいずれかに記載の塩基配列からなる DNAが好ましぐ配列番号： 1 、 3〜6、 14、 16、 17、 22または 25のいずれかに記載の塩基配列からなる DNAがよ り好まし 、が、これらに限定されるものではな！/、。  “DNA that consists of the base sequence described in IJ numbers: 1-6, 8, 14, 16, 17, 19-22, 24, 25, 28 or 30! SEQ ID NO: 1-6, 14, 16, 17, 19, 20, 22, 25, or 30 DNA having a nucleotide sequence described in any one of SEQ ID NOs: 1, 3-6, 14, 16 More preferably, the DNA comprising the nucleotide sequence described in any one of, 17, 22 or 25 is not limited to these! /.
また本発明においては、以下の（7)〜（10)の場合に、植物の榭幹木部が、木繊維 形成が盛んな木部であると判定される。さらに、（8)または（9)の場合に、植物の榭幹 木部が、同様な条件で採取されたグロプルスの樹幹の木部と比較して、木繊維長は 短ぐ直径は細ぐ細胞壁は薄ぐフィブリル傾角は急で、木繊維率と比重は低い、と V、うパルプ特性を有する木繊維が形成されて!ヽる木部、または将来的に該パルプ特 性を有する木繊維が形成される木部であると判定される。また、（10)の場合に、植物 の榭幹木部が、同様な条件で採取されたカマルドレンシス榭幹の木部と比較して、木 繊維長は長ぐ直径は太ぐ細胞壁は厚ぐフィブリル傾角は緩ぐ木繊維率と比重は 高い、という優れたパルプ特性を有する木繊維が形成されている木部、または将来 的に該パルプ特性を有する木繊維が形成される木部であると判定される。  Further, in the present invention, in the following cases (7) to (10), it is determined that the trunk trunk portion of the plant is a xylem portion in which wood fiber formation is active. Furthermore, in the case of (8) or (9), the length of the fiber of the trunk of the plant is shorter and the diameter of the cell wall is narrower than that of the grops of the trunk of the groprus collected under the same conditions. The fibril inclination angle is steep, the wood fiber ratio and specific gravity are low, and V, a wood fiber having a pulp characteristic is formed! It is determined that the xylem is formed. In the case of (10), the length of the tree fiber is longer and the cell wall is thicker than the length of the stem of the stem of the plant, compared to the xylem of the Camaldrensis trunk collected under the same conditions. It is a xylem where wood fibers with excellent pulp properties are formed, such as the ratio of loose fibrils is high and the specific gravity is high, or the xylem where wood fibers with the pulp properties are formed in the future It is determined.
また、本発明の検査方法を利用することで材質の量的 ·質的な事前予測が可能とな る。また、植林事業において、生長状態をもとに適切な施業 (除草'間伐等)を適切な 時期に施すことができる。例えば、本発明におけるユーカリ属カマルドレンシス榭幹 の師部と木部での発現パターンと本発明の検査方法で得られた発現パターンに相 関があれば (好ましくはピアソンの相関係数 0.5以上、より好ましくは 0.7以上、以下同 じ)、被検榭幹木部に、質的には低レベルな木繊維 (低品質パルプ:短繊維、直径は 細ぐ細胞壁は薄ぐフィブリル傾角は急で、低木繊維率と低比重）が形成されている 、もしくは将来的に形成される状態であり、木繊維量 (容積重)も少ないもしくは将来 的に少なくなる（平均的な容積重 450〜500kg/m3以下）という予測ができる。一方、本 発明におけるユーカリ属グロブルス榭幹の師部と木部での発現パターンと本発明の 検査方法で得られた発現パターンに相関があれば、被検榭幹木部に、質的には優 れた繊維 (高品質パルプ:長繊維、直径は太ぐ細胞壁は厚ぐフィブリル傾角は緩く 、高木繊維率と高比重）が形成されている、もしくは将来的に形成される状態であり、 木繊維量 (容積重)も多 、もしくは将来的に多くなる（平均的な容積重 450〜500kg/m 3以上）という予測ができる。さらに、上記のような材質予測により、植物の生長状態の 善し悪しが明ら力となるため、悪いと判断されれば、貧栄養地では施肥を行ったり、 雑草が茂っていれば除草作業を早期に行ったり、植栽密度が高ければ間伐を行つ たり、といった施業を迅速かつ適切に行うことが可能になる。現在、前述のような材質 予測や施業方針の決定は、多分に現場の経験や勘、もしくは事後の化学分析等に 頼っており、本願で示すような事前に判別できる科学的指標は無い。 In addition, by using the inspection method of the present invention, quantitative and qualitative advance prediction of materials can be performed. In afforestation projects, appropriate operations (weeding, thinning, etc.) can be carried out at appropriate times based on the growth conditions. For example, if there is a correlation between the expression pattern in the phloem and xylem of Eucalyptus genus Camaldrensis trunk in the present invention and the expression pattern obtained by the test method of the present invention (preferably Pearson's correlation coefficient of 0.5 or more , More preferably 0.7 or more, and the same below), qualitatively low level of wood fiber (low-quality pulp: short fiber, thin diameter, thin cell wall, thin fibril tilt angle) , Shrub fiber rate and low specific gravity) are formed It can also be predicted that it will be formed in the future, and the amount of wood fibers (volume weight) will be small or will be small in the future (average volume weight 450 to 500 kg / m 3 or less). On the other hand, if there is a correlation between the expression pattern in the phloem and xylem of the Eucalyptus globulus trunk in the present invention and the expression pattern obtained by the test method of the present invention, Excellent fibers (high quality pulp: long fibers, thicker cell walls are thicker, fibril inclination is gentle, high wood fiber ratio and high specific gravity) are formed, or are formed in the future. It can be predicted that the amount of fiber (volume weight) will be large or will increase in the future (average weight of 450 to 500 kg / m 3 or more). In addition, the material quality prediction as described above makes it clear that the state of plant growth is good or bad, so if it is judged to be bad, fertilization is performed in an oligotrophic area, or if weeds are overgrown, early weeding work is performed. If the planting density is high, thinning can be performed quickly and appropriately. At present, material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance.
(7)植物の榭幹 (好ましくは榭幹基部または榭幹中央部）の師部側および木部側に おいて、「ユーカリ属カマルドレンシスおよびユーカリ属グロブルスの榭幹において、 師部側と比較して木部側で発現レベルが上昇している DNA」のうち少なくとも 1つの D NAの発現レベルを検出し、師部側および木部側における該 DNAの発現レベルを比 較した結果、植物の榭幹における該 DNAの発現レベル力 師部側と比較して木部側 で上昇している場合 (7) At the phloem side and the xylem side of the trunk of the plant (preferably the trunk trunk or the middle of the trunk), “In the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules, As a result of detecting the expression level of at least one DNA out of `` DNA whose expression level is increased on the xylem side compared with the other '', the expression level of the DNA on the phloem side and the xylem side was compared. The level of expression of the DNA in the trunk of the tree is higher on the xylem side than on the phloem side
本発明にお 、て、「ユーカリ属カマルドレンシスおよびユーカリ属グロブルスの榭幹 において、師部側と比較して木部側で発現レベルが上昇している DNA」としては、以 下の（a)〜（e)の!、ずれかに記載の DNAが挙げられる。  In the present invention, “DNA having an increased expression level on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules” includes the following (a ) To (e)!
(a)配列番号： 45、 46、 51、 55、 56または 64のいずれかに記載のアミノ酸配列から なるタンパク質をコードする DNA  (a) DNA encoding a protein comprising the amino acid sequence set forth in any of SEQ ID NOs: 45, 46, 51, 55, 56 or 64
(b)配列番号： 11、 12、 17、 21、 22または 30のいずれかに記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 11, 12, 17, 21, 22, or 30
(c)配列番号： 45、 46、 51、 55、 56または 64のいずれかに記載のアミノ酸配列から なるタンパク質と 50%以上の相同性を有するタンパク質をコードする DNA (d)配列番号： 11、 12、 17、 21、 22または 30のいずれかに記載の塩基配列からな る DNAとストリンジェントな条件下でハイブリダィズする DNA (c) DNA encoding a protein having 50% or more homology with a protein consisting of the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, 56 or 64 (d) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 11, 12, 17, 21, 22, or 30
(e)酉己歹 IJ番号： 45、 46、 51、 55、 56また ίま 64の! /、ずれ力に記載のアミノ酸酉己歹 IJにお いて 1または複数のアミノ酸が置換、欠失、付加、および Ζまたは挿入されたアミノ酸 配列からなるタンパク質をコードする DNA  (e) IJ number: 45, 46, 51, 55, 56 or ί or 64! /, amino acids listed in the displacement force DNA that encodes a protein consisting of an amino acid sequence that is added and inserted or trapped
「配列番号： 11、 12、 17、 21、 22または 30のいずれかに記載の塩基配列力 なる DNA」としては、配列番号： 11、 12、 17、 22または 30のいずれかに記載の塩基配列 力 なる DNAが好ましぐ配列番号： 11、 12、 17または 22のいずれかに記載の塩基 配列からなる DNAがより好ましいが、これらに限定されるものではない。  The “DNA having the nucleotide sequence described in any of SEQ ID NO: 11, 12, 17, 21, 22 or 30” includes the nucleotide sequence described in any of SEQ ID NO: 11, 12, 17, 22 or 30. DNA consisting of the base sequence described in any one of SEQ ID NOs: 11, 12, 17 or 22 is more preferred, but is not limited thereto.
(8)植物の榭幹 (好ましくは榭幹基部または榭幹中央部）の師部側および木部側に おいて、「ユーカリ属カマルドレンシスの榭幹において、師部側と比較して木部側で 発現レベルが上昇し、ユーカリ属グロブルスの榭幹において、師部側と比較して木部 側で発現レベルが減少して 、る DNA」のうち少なくとも 1つの DNAの発現レベルを検 出し、師部側および木部側における該 DNAの発現レベルを比較した結果、植物の榭 幹における該 DNAの発現レベル力 師部側と比較して木部側で上昇して 、る場合 本発明において、「ユーカリ属カマルドレンシスの榭幹において、師部側と比較して 木部側で発現レベルが上昇し、ユーカリ属グロブルスの榭幹において、師部側と比 較して木部側で発現レベルが減少して!/ヽる DNA」としては、以下の（a)〜（e)の!ヽず れかに記載の DNAが挙げられる。 (8) At the phloem side and the xylem side of the trunk of the plant (preferably the trunk trunk or the middle of the trunk), “In the trunk of the Eucalyptus genus Camaldrensis, the tree compared to the phloem side. The level of expression increases on the part side, and the expression level of eucalyptus globulus trunks is reduced on the xylem side compared to the phloem side, and the expression level of at least one DNA is detected. In the present invention, as a result of comparing the expression level of the DNA on the phloem side and the xylem side, the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side. , “In the trunk of Eucalyptus genus Camaldrensis, the expression level increased on the xylem side compared to the phloem side, and in the trunk of the Eucalyptus globules, the expression level on the xylem side compared to the phloem side Levels decrease! / Speaking DNA ”includes the following (a) to (e)! Not Re include a DNA according to any.
(a)配列番号： 42、 47、 53、 57〜59または 67のいずれかに記載のアミノ酸配列から なるタンパク質をコードする DNA  (a) DNA encoding a protein comprising the amino acid sequence set forth in any of SEQ ID NOs: 42, 47, 53, 57-59 or 67
(b)配列番号： 8、 13、 19、 23〜25または 33のいずれかに記載の塩基配列を含む D NA  (b) SEQ ID NO: 8, 13, 19, 23 to 25 or 33 containing the nucleotide sequence described in 33
(c)配列番号： 42、 47、 53、 57〜59または 67のいずれかに記載のアミノ酸配列から なるタンパク質と 50%以上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having a homology of 50% or more with a protein consisting of the amino acid sequence of SEQ ID NO: 42, 47, 53, 57-59 or 67
(d)配列番号： 8、 13、 19、 23〜25または 33のいずれかに記載の塩基配列力もなる DNAとストリンジェントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with the DNA of any one of SEQ ID NOs: 8, 13, 19, 23-25 or 33
(e)配列番号： 42、 47、 53、 57〜59または 67のいずれかに記載のアミノ酸配列に おいて 1または複数のアミノ酸が置換、欠失、付加、および/または挿入されたァミノ 酸配列からなるタンパク質をコードする DNA (e) the amino acid sequence of SEQ ID NO: 42, 47, 53, 57-59 or 67 DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted
「配列番号： 8、 13、 19、 23〜25または 33のいずれかに記載の塩基配列からなる DNA」としては、配列番号： 8、 13、 19、 24、 25または 33のいずれかに記載の塩基配 列からなる DNAが好ましぐ配列番号： 13、 19または 25のいずれかに記載の塩基配 列からなる DNAがより好ましいが、これらに限定されるものではない。  "DNA consisting of the base sequence described in any of SEQ ID NO: 8, 13, 19, 23-25 or 33" is the sequence described in any of SEQ ID NO: 8, 13, 19, 24, 25 or 33 DNA consisting of a base sequence is preferred, but DNA consisting of the base sequence described in any one of SEQ ID NOS: 13, 19, or 25 is more preferred, but is not limited thereto.
(9)植物の榭幹 (好ましくは榭幹基部または榭幹中央部）の師部側および木部側に おいて、「ユーカリ属カマルドレンシスの榭幹において、師部側と比較して木部側で 発現レベルが上昇し、ユーカリ属グロブルスの榭幹において、師部側と木部側で発 現レベルが同等な DNA」のうち少なくとも 1つの DNAの発現レベルを検出し、師部側 および木部側における該 DNAの発現レベルを比較した結果、植物の榭幹における 該 DNAの発現レベル力 師部側と比較して木部側で上昇して 、る場合 (9) On the phloem side and the xylem side of the trunk of the plant (preferably the trunk trunk or the middle of the trunk), “In the trunk of the Eucalyptus genus Camaldrensis, the tree compared to the phloem side. The expression level rises on the side, and in the trunk of Eucalyptus globules, the expression level of at least one DNA of `` DNA with the same expression level on the phloem side and the xylem side '' is detected. When the expression level of the DNA on the xylem side is compared, the expression level of the DNA in the trunk of the plant is higher on the xylem side than on the mentor side
本発明において、「ユーカリ属カマルドレンシスの榭幹において、師部側と比較して 木部側で発現レベルが上昇し、ユーカリ属グロブルスの榭幹において、師部側と木 部側で発現レベルが同等な DNA」としては、以下の（a)〜（e)のいずれかに記載の D NAが挙げられる。  In the present invention, the expression level is increased on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis, and the expression level on the phloem side and xylem side in the trunk of the Eucalyptus globules Examples of the DNA having the same DNA include DNAs described in any of the following (a) to (e).
(a)酉己歹 IJ番号： 35〜40、 43、 48、 50、 54、 61、 62また ίま 66の!/、ずれ力に記載のァ ミノ酸配列力 なるタンパク質をコードする DNA  (a) IJ number: 35-40, 43, 48, 50, 54, 61, 62 or ί or 66! /, DNA encoding the protein with amino acid alignment ability described in the displacement force
(b)酉己歹 IJ番号： 1〜6、 9、 14、 16、 20、 27、 28また ίま 32の!/、ずれ力に記載の塩基酉己 列を含む DNA  (b) IJ numbers: 1 to 6, 9, 14, 16, 20, 27, 28 or ί or 32! /, DNA containing the base sequence described in the displacement force
(c)酉己歹 IJ番号： 35〜40、 43、 48、 50、 54、 61、 62また ίま 66の!/、ずれ力に記載のァ ミノ酸配列力 なるタンパク質と 50%以上の相同性を有するタンパク質をコードする D ΝΑ  (c) IJ number: 35-40, 43, 48, 50, 54, 61, 62 or ί or 66! /, amino acid alignment ability described in the displacement force 50% or more homology with the protein D コ ー ド which encodes a protein with sex
(d)酉己歹 IJ番号： 1〜6、 9、 14、 16、 20、 27、 28また ίま 32の!/、ずれ力に記載の塩基酉己 列からなる DNAとストリンジェントな条件下でハイブリダィズする DNA  (d) IJ numbers: 1 to 6, 9, 14, 16, 20, 27, 28 or ί or 32! /, DNA consisting of the base sequence described in the displacement force and stringent conditions DNA hybridizing with
(e)酉己歹 IJ番号： 35〜40、 43、 48、 50、 54、 61、 62また ίま 66の! /、ずれ力に記載のァ ミノ酸配列において 1または複数のアミノ酸が置換、欠失、付加、および Ζまたは挿 入されたアミノ酸配列力 なるタンパク質をコードする DNA 「酉己歹 IJ番号： 1〜6、 9、 14、 16、 20、 27、 28また ίま 32の!ヽずれ力に記載の塩基酉己 列からなる DNA」としては、配列番号： 1〜6、 9、 14、 16、 20または 27のいずれかに 記載の塩基配列力もなる DNAが好ましぐ配列番号： 1、 3〜6、 14または 16のいず れかに記載の塩基配列力 なる DNAがより好ましいが、これらに限定されるものでは ない。 (e) 酉 己 ： IJ number: 35-40, 43, 48, 50, 54, 61, 62 or ί or 66! /, one or more amino acids are substituted in the amino acid sequence described in shear force, Deletion, addition, and Ζ or inserted amino acid DNA “DNA consisting of the base sequence described in the IJ number: 1-6, 9, 14, 16, 20, 27, 28 or 32! DNA sequence ability described in any of 6, 9, 14, 16, 20, or 27 is also preferred by DNA. SEQ ID NO: 1, 3, 6, 14, or 16 Although DNA is more preferable, it is not limited to these.
[0065] (10)植物の榭幹 (好ましくは榭幹基部）の師部側および木部側にお 、て、上記「ュ 一力リ属カマルドレンシスの樹幹にぉ 、て、師部側と比較して木部側で発現レベルが 上昇し、ユーカリ属グロブルスの榭幹において、師部側と比較して木部側で発現レべ ルが減少している DNA」のうち少なくとも 1つの DNAの発現レベルを検出し、師部側 および木部側における該 DNAの発現レベルを比較した結果、植物の榭幹における 該 DNAの発現レベル力 師部側と比較して木部側で減少して 、る場合  [0065] (10) On the phloem side and xylem side of the trunk (preferably trunk base) of the plant, on the phloem side above the trunk of the above-mentioned genus Camaldrensis At least one of the DNAs whose expression level is increased on the xylem side compared to the eucalyptus globulus and whose expression level is decreased on the xylem side compared to the phloem side As a result of detecting the expression level of the DNA and comparing the expression level of the DNA on the phloem side and the xylem side, the expression level of the DNA in the trunk of the plant decreased on the xylem side compared to the phloem side If
さらに、本発明においては、異なる 2つの植物 (好ましくはユーカリ）の榭幹木部の 生長状態 (パルプ品質)を検査する方法を提供する。すなわち、異なる 2つの植物の 榭幹木部うち、どちらが優れたパルプ品質なのか、または劣るのかを検査する方法を 提供する。この方法は、複数の植物の榭幹木部から、優れた、または劣った榭幹木 部を選別することにも使用できる。  Furthermore, the present invention provides a method for inspecting the growth state (pulp quality) of the stem portion of two different plants (preferably eucalyptus). In other words, it provides a method for inspecting which of two different plant trunks is superior or inferior in pulp quality. This method can also be used to select good or inferior trunk trunks from multiple plant trunk trunks.
より具体的には、以下の（11)または（12)の場合に、異なる 2つの植物の榭幹木部 のうち、片方の植物（以下、植物 Aと称す)の榭幹木部と比較して他方の植物（以下、 植物 Bと称す)の榭幹木部は、優れたパルプ品質を有する木部であり、榭幹木部 Bと 比較して榭幹木部 Aは、劣ったパルプ品質を有する木部であると判定される。従って 、以下の（11)または（12)は、例えば育種現場において、エリート木やプラスツリーと 呼ばれる優良系統を選抜するときの指標になる。現在、エリート木等の選抜は、榭高 や直径などの外見上の数値や伐採後の材質分析値を指標に行われており、上記指 標で選抜する技術 (概念)は無 、。  More specifically, in the case of (11) or (12) below, it is compared with the trunk trunk of one of the two different plants (hereinafter referred to as plant A). The stem of the other plant (hereinafter referred to as plant B) is a xylem with excellent pulp quality. Compared to the stem B, B It is determined that the xylem has Therefore, the following (11) or (12) is an index for selecting an excellent line called an elite tree or a plus tree at a breeding site, for example. At present, selection of elite trees, etc., is based on apparent values such as height and diameter, and material analysis values after cutting. There is no technology (concept) to select with the above indicators.
[0066] (11)異なる 2つの植物の榭幹木部 (好ましくは榭幹基部または榭幹中央部の木部） にお 、て、「ユーカリ属グロブルスの樹幹の木部側と比較してユーカリ属カマルドレン シスの榭幹の木部側で発現レベルが上昇している DNA」のうち少なくとも 1つの DNA の発現レベルを検出し、異なる 2つの植物の榭幹木部における該 DNAの発現レベル を比較した結果、該 DNAの発現レベル力 植物 Bと比較して植物 Aで上昇している場 本発明にお 、て、 「ユーカリ属グロブルスの樹幹の木部側と比較してユーカリ属カ マルドレンシスの樹幹の木部側で発現レベルが上昇している DNA」としては、以下の (a)〜（e)の!、ずれかに記載の DNAが挙げられる。 [11] (11) In the trunk trunk of two different plants (preferably the trunk of the trunk trunk or the middle of the trunk), the eucalyptus is compared with the xylem side of the trunk of the Eucalyptus globulus. The expression level of at least one of the DNAs whose expression level is increased on the xylem side of the trunk of the genus Camaldrensis is detected, and the expression level of the DNA in the trunk xylem of two different plants As a result of the comparison, the expression level of the DNA is higher in the plant A than in the plant B. In the present invention, the Eucalyptus genus Camaldrensis is compared with the xylem side of the trunk of the Eucalyptus globules. Examples of “DNA having an increased expression level on the xylem side of the trunk” include the following DNAs (a) to (e)!
(a)配列番号: 45、 46、 51または 56のいずれかに記載のアミノ酸配列からなるタン パク質をコードする DNA  (a) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 45, 46, 51 or 56
(b)配列番号： 11、 12、 17または 22のいずれかに記載の塩基配列を含む DNA (b) DNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 11, 12, 17 or 22
(c)配列番号: 45、 46、 51または 56のいずれかに記載のアミノ酸配列からなるタン ノ ク質と 50%以上の相同性を有するタンパク質をコードする DNA (c) DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence of SEQ ID NO: 45, 46, 51 or 56
(d)配列番号： 11、 12、 17または 22のいずれかに記載の塩基配列からなる DNAとス トリンジェントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in any of SEQ ID NOS: 11, 12, 17 or 22
(e)配列番号： 45、 46、 51または 56のいずれかに記載のアミノ酸配列において 1ま たは複数のアミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配列か らなるタンパク質をコードする DNA  (e) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence of SEQ ID NO: 45, 46, 51 or 56 DNA to encode
「配列番号： 11、 12、 17または 22のいずれかに記載の塩基配列からなる DNA」とし ては、配列番号： 12または 17に記載の塩基配列からなる DNAが好ましぐ配列番号: 12のいずれかに記載の塩基配列力 なる DNAがより好ましいが、これらに限定される ものではない。  “DNA consisting of the nucleotide sequence set forth in any of SEQ ID NO: 11, 12, 17 or 22” is preferably the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 12 or 17 Any of the DNAs having base sequence strengths described in any one of these is more preferable, but not limited thereto.
(12)異なる 2つの植物の榭幹木部 (好ましくは榭幹基部または榭幹中央部の木部） にお 、て、「ユーカリ属グロブルスの樹幹の木部側と比較してユーカリ属カマルドレン シスの榭幹の木部側で発現レベルが減少して 、る DNA」のうち少なくとも 1つの DNA の発現レベルを検出し、異なる 2つの植物の榭幹木部における該 DNAの発現レベル を比較した結果、該 DNAの発現レベル力 植物 Bと比較して植物 Aで減少している場 本発明にお 、て、 「ユーカリ属グロブルスの樹幹の木部側と比較してユーカリ属カ マルドレンシスの樹幹の木部側で発現レベルが減少している DNA」としては、以下の (a)〜（e)の!、ずれかに記載の DNAが挙げられる。 (a)配列番号： 55に記載のアミノ酸配列力 なるタンパク質をコードする DNA (12) In the trunk trunk of two different plants (preferably the trunk of the trunk trunk or the middle of the trunk), the eucalyptus genus Camaldrensis is compared with the xylem side of the trunk of the Eucalyptus globulus. The expression level of at least one DNA of `` DNA with decreased expression level at the xylem of the trunk of the tree '' was detected, and the expression level of the DNA in the trunk of the two different plants was compared. In the present invention, the expression level of the DNA is reduced in the plant A compared with the plant B. In the present invention, the tree of the Eucalyptus genus Camaldlensis is compared with the tree side of the Eucalyptus globulus. Examples of the “DNA whose expression level is reduced on the part side” include the DNAs described in (a) to (e) below, or any of them. (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 55
(b)配列番号： 21に記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 21
(c)配列番号： 55に記載のアミノ酸配列力 なるタンパク質と 50%以上の相同性を有 するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in SEQ ID NO: 55
(d)配列番号： 21に記載の塩基配列力もなる DNAとストリンジェントな条件下でノ、イブ リダィズする DNA  (d) DNA that has the nucleotide sequence of SEQ ID NO: 21 and DNA that can be hybridized under stringent conditions
(e)配列番号： 55に記載のアミノ酸配列において 1または複数のアミノ酸が置換、欠 失、付加、および Zまたは挿入されたアミノ酸配列からなるタンパク質をコードする DN A  (e) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence set forth in SEQ ID NO: 55
[0068] 本発明の検査方法を利用することで、上記（1)〜（12)のいずれかに該当する木部 または該木部を有する植物を選択し、選択された木部または該木部を有する植物か ら均一なパルプを作製することが可能であり、パルプ製造の効率化、コストの削減、 新規な特性を持った紙の製造が可能となる。なお、以下の（1)〜（12)のいずれかに 該当する木部または該木部を有する植物を選択後、当業者に周知の方法に従って、 選択された木部間または該木部を有する植物間で木繊維状態が均一であるかを確 認した後に、パルプを作製することもできる。また、以下の（1)〜（12)のいずれかに 該当しな 、木部または該木部を有する植物を集めて、任意の配合のパルプを作製 することちでさる。  [0068] By using the inspection method of the present invention, a xylem corresponding to any of the above (1) to (12) or a plant having the xylem is selected, and the selected xylem or the xylem It is possible to produce a uniform pulp from plants that have a large amount of pulp, making it possible to increase the efficiency of pulp production, reduce costs, and produce paper with new characteristics. In addition, after selecting a xylem corresponding to any of the following (1) to (12) or a plant having the xylem, according to a method well known to those skilled in the art, between the selected xylems or having the xylem Pulp can also be produced after confirming whether the wood fiber state is uniform among plants. In addition, if it does not fall under any of the following (1) to (12), it is obtained by collecting a xylem or a plant having the xylem to produce a pulp having an arbitrary composition.
[0069] 本発明はまた、植物の転写因子をコードする DNAとプロモーター DNAを提供する。  [0069] The present invention also provides a DNA encoding a plant transcription factor and a promoter DNA.
本発明における植物の転写因子をコードする DNAは、植物の木繊維細胞形成の 制御に利用できると考えられる。一方、該 DNAのプロモーター DNAは、木繊維組織あ るいは木繊維形成時期特異的発現の制御に利用できると考えられる。また、植物榭 幹の木繊維細胞形成を制御する機能を有するタンパク質をコードする DNAの発現（ レベル)情報は、植物体の生長状態や木繊維形成状態、材質の量的'質的な事前予 測に利用できると考えられる。従って、これらを用いることで、人為的な木繊維細胞形 態形成の改変や任意の遺伝子 ·タンパク質の木繊維細胞特異的発現、植物体の生 長状態や木繊維形成状態、材質の量的'質的な事前予測に利用することができると 考えられる。その結果、パルプ製造の効率化、コストの削減、新規な特性を持った紙 の製造が可能になる。 It is considered that DNA encoding a plant transcription factor in the present invention can be used to control the formation of plant fiber fibers. On the other hand, it is considered that the promoter DNA of the DNA can be used to control expression specific to wood fiber tissue or wood fiber formation time. In addition, the expression (level) information of DNA encoding a protein having a function to control the formation of plant fiber in the tree trunk is based on the quantitative state of the plant growth state, tree fiber formation state, and material quality. It can be used for measurement. Therefore, by using these, artificial modification of tree fiber cell shape formation, tree fiber cell-specific expression of any gene / protein, plant growth state, tree fiber formation state, quantitative quantity of materials It can be used for qualitative advance prediction. As a result, more efficient pulp production, reduced costs, and paper with new characteristics Can be manufactured.
[0070] 植物において木繊維形成を制御することは、工業や農業の分野において、様々な 重要な意義を有する。例えば、ユーカリ属カマルドレンシス種の木繊維性質の改変 は、繊維長を長くすることによりパルプ等繊維原材料の繊維特性の向上などの点で 有意義である。またユーカリ属グロブルス種のセルロース ·へミセルロース含量を高め ることによるパルプ等の収量増大や、蒸解効率の向上などをもたらし、経済性や収益 性の点で有意義である。  [0070] Controlling wood fiber formation in plants has various important implications in the fields of industry and agriculture. For example, modification of the wood fiber properties of Eucalyptus genus Camaldrensis is significant in terms of improving the fiber properties of fiber raw materials such as pulp by increasing the fiber length. In addition, increasing the cellulose and hemicellulose content of Eucalyptus globulae species increases the yield of pulp and the like, and improves the cooking efficiency, which is significant in terms of economy and profitability.
[0071] 本発明の DNAの由来する植物としては、特に制限はなぐ例えば、穀類、野菜、果 榭等の有用農作物 (飼料作物を含む)、パルプ等の繊維原材料植物、観葉植物等の 鑑賞用植物等が挙げられる。該植物としては特に制限はなぐユーカリ、マツ、ァカシ ァ、ポプラ、スギ、ヒノキ、タケ、ィチイ、イネ、トウモロコシ、コムギ、ォォムギ、ライムギ 、ジャガイモ、タバコ、サトウダイコン、サトウキビ、ナタネ、ダイズ、ヒマヮリ、ヮタ、ォレ ンジ、ブドウ、モモ、ナシ、リンゴ、トマト、ハクサイ、キャベツ、ダイコン、ニンジン、カボ チヤ、キユウリ、メロン、パセリ、ラン、キク、ユリ、サフラン等を例示することができる。  [0071] The plant from which the DNA of the present invention is derived is not particularly limited, for example, useful crops (including forage crops) such as cereals, vegetables and fruit straw, fiber raw material plants such as pulp, ornamental plants, etc. Examples include plants. There are no particular restrictions on the plant, such as eucalyptus, pine, acacia, poplar, cedar, cypress, bamboo, yew, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower, Examples include ivy, orange, grape, peach, pear, apple, tomato, Chinese cabbage, cabbage, Japanese radish, carrot, cabbage, cucumber, melon, parsley, orchid, chrysanthemum, lily and saffron.
[0072] 本発明にお!/、て、植物の転写因子をコードする DNAとしては、以下の（a)〜（e)の いずれかに記載の DNAが挙げられる。  [0072] In the present invention, examples of the DNA encoding a plant transcription factor include the DNAs described in any of the following (a) to (e).
(a)配列番号： 112〜 122の 、ずれかに記載のアミノ酸配列力もなるタンパク質をコ ードする DNA  (a) DNA encoding a protein having an amino acid sequence ability described in any one of SEQ ID NOs: 112 to 122
(b)配列番号：101〜： L 11の!、ずれかに記載の塩基配列を含む DNA  (b) SEQ ID NO: 101-: DNA comprising the base sequence described in L11!
(c)配列番号： 112〜 122の 、ずれかに記載のアミノ酸配列力もなるタンパク質と 50 %以上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein of SEQ ID NO: 112 to 122, which also has an amino acid sequence ability described in any one of
(d)配列番号： 101〜111のいずれかに記載の塩基配列からなる DNAとストリンジ ントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 101-111
(e)配列番号： 112〜 122の 、ずれかに記載のアミノ酸配列にお 、て 1または複数の アミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配列力 なるタンパ ク質をコードする DNA  (e) In the amino acid sequence according to any one of SEQ ID NOS: 112 to 122, one or a plurality of amino acids are substituted, deleted, added, and encoded by a protein having an amino acid sequence ability by Z or insertion DNA
[0073] 本発明において、ストリンジェントなハイブリダィゼーシヨン条件としては、 O.lxSSC溶 液中にて、 60°C、ー晚放置の条件またはこれと同等のストリンジエンシーのノ、イブリダ ィゼーシヨン条件が挙げられる。このような条件において、配列番号： 101〜111のい ずれかに記載の塩基配列力もなる DNAとハイブリダィズする DNAが単離できる。 [0073] In the present invention, stringent hybridization conditions include conditions of standing at 60 ° C in an O.lxSSC solution, or a stringency equivalent to this, An example is the condition. Under such conditions, DNA that hybridizes with DNA having the nucleotide sequence described in any one of SEQ ID NOs: 101 to 111 can be isolated.
[0074] 具体的には、植物より、 DNAを抽出し、遺伝子ライブラリーを構築し、同様の条件に よりスクリーニングを行うか、もしくは、抽出した DNAに対して、配列番号： 101〜： L 11 のいずれかに記載の塩基配列より、任意の 20mer程度の配列をプライマーに用いて 、劉らによって確立された TAIL-PCR法により、連続する近傍配列を容易に取得でき る。 [0074] Specifically, DNA is extracted from a plant, a gene library is constructed, and screening is performed under the same conditions, or the extracted DNA is subjected to SEQ ID NO: 101-: L 11 From the base sequence described in any of the above, a contiguous neighboring sequence can be easily obtained by the TAIL-PCR method established by Liu et al. Using an arbitrary 20-mer sequence as a primer.
[0075] また、本発明は、配列番号： 112〜122のいずれかに記載のアミノ酸配列力もなる タンパク質と 50%以上の相同性を有するタンパク質をコードする DNAも提供する。こ のような DNAは、実施例に記載の構造的特徴を有するタンパク質をコードする DNAで あることが好ましい。  [0075] The present invention also provides a DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in any of SEQ ID NOs: 112 to 122. Such DNA is preferably DNA encoding a protein having the structural characteristics described in the Examples.
[0076] 配列番号： 112〜122のいずれかに記載のアミノ酸配列からなるタンパク質と 50% 以上の相同性を有するタンパク質をコードする DNAは、当業者においては、一般的 に公知の方法により単離することが可能である。例えば、ハイブリダィゼーシヨン技術 (Southern, EM., J Mol Biol, 1975, 98, 503.)やポリメラーゼ連鎖反応（PCR)技術（Sai ki, RK. et al, Science, 1985, 230, 1350.、 Saiki, RK. et al, Science 1988, 239, 487.) を利用する方法が挙げられる。すなわち、配列番号： 101〜： L 11のいずれかに記載 の塩基配列力もなる DNAもしくはその一部をプローブとして、また、配列番号： 101〜 111の 、ずれかに記載の塩基配列力もなる DNAに特異的にハイブリダィズするオリ ゴヌクレオチドをプライマーとして、植物から配列番号： 101〜111のいずれかに記載 の塩基配列力 なる DNAと高 、相同性を有する DNAを単離することは、当業者にとつ て通常行 ヽ得ることである。  [0076] A DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence of SEQ ID NO: 112 to 122 is generally isolated by a person skilled in the art by a known method. Is possible. For example, hybridization technology (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR) technology (Sai ki, RK. Et al, Science, 1985, 230, 1350. Saiki, RK. Et al, Science 1988, 239, 487.). That is, DNA having a base sequence ability described in any of SEQ ID NOs: 101 to L11 or a part thereof as a probe, or a DNA having a base sequence ability described in any of SEQ ID NOs: 101 to 111 It is known to those skilled in the art to isolate a DNA having high homology with a DNA having a nucleotide sequence described in any one of SEQ ID NOS: 101 to 111 from a plant using an oligonucleotide that specifically hybridizes as a primer. It is about normal deeds.
[0077] このような DNAを単離するためには、好ましくはストリンジヱントな条件下でハイブリ ダイゼーシヨン反応を行う。本発明にお 、てストリンジェントなハイブリダィゼーシヨン 条件とは、 6M尿素、 0.4% SDS、 0.5 X SSCの条件またはこれと同等のストリンジェン シ一のハイブリダィゼーシヨン条件を指す。よりストリンジエンシーの高い条件、例えば 、 6M尿素、 0.4% SDS、 0.1 X SSCの条件下では、より相同性の高い DNAを単離でき ると期待される。こうして単離された DNAは、アミノ酸レベルにおいて配列番号： 101 〜111のいずれかに記載の塩基配列力もなる DNAからコードされるアミノ酸配列と高 い相同性を有すると考えられる。高い相同性とは、アミノ酸配列全体で少なくとも 50% 以上、好ましくは 70%以上、より好ましくは 80%以上、さらに好ましくは 90%以上、さら により好ましくは 95%以上、最も好ましくは 98%以上の配列の同一性を指す。 [0077] In order to isolate such a DNA, a hybridization reaction is preferably performed under stringent conditions. In the present invention, stringent hybridization conditions refer to conditions of 6M urea, 0.4% SDS, 0.5 X SSC or equivalent stringency hybridization conditions. It is expected that DNA with higher homology can be isolated under conditions with higher stringency, for example, 6M urea, 0.4% SDS, and 0.1X SSC. The DNA thus isolated has the SEQ ID NO: 101 at the amino acid level. It is considered to have a high homology with an amino acid sequence encoded by DNA having the nucleotide sequence ability described in any of ˜111. High homology means at least 50% or more of the entire amino acid sequence, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, more preferably 95% or more, and most preferably 98% or more. Refers to sequence identity.
[0078] アミノ酸配列や塩基配列の同一性は、カーリンおよびアルチユールによるアルゴリズ ム BLAST (Proc. Natl. Acad. Sei. USA, 1990, 87, 2264-2268.、 Karlin, S. & Altschul, SF" Proc. Natl. Acad. Sei. USA, 1993, 90, 5873.)を用いて決定できる。 BLASTのァ ルゴリズムに基づ 、た BLASTNや BLASTXと呼ばれるプログラムが開発されて!、る (A1 tschul, SF. et al.， J Mol Biol, 1990, 215, 403.) 0 BLASTNを用いて塩基配列を解析 する場合は、パラメータ一は、例えば score= 100、 wordlength= 12とする。また、 BLA STXを用いてアミノ酸配列を解析する場合は、パラメータ一は、例えば score = 50、 wo rdlength=3とする。 BLASTと Gapped BLASTプログラムを用いる場合は、各プログラム のデフォルトパラメーターを用いる。これらの解析方法の具体的な手法は公知である (http://www.ncbi.nlm.nih.gov/)。 [0078] The identity of the amino acid sequence and nucleotide sequence is determined by the algorithm BLAST (Proc. Natl. Acad. Sei. USA, 1990, 87, 2264-2268., Karlin, S. & Altschul, SF "Proc. Natl. Acad. Sei. USA, 1993, 90, 5873.) Based on the BLAST algorithm, programs called BLASTN and BLASTX have been developed !, and (A1 tschul, SF. et al., J Mol Biol, 1990, 215, 403.) 0 When analyzing the base sequence using BLASTN, the parameter 1 is, for example, score = 100, wordlength = 12, and BLA STX When analyzing an amino acid sequence, the parameter 1 is, for example, score = 50 and word length = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. The technique is known (http://www.ncbi.nlm.nih.gov/).
[0079] また、本発明の DNAには、配列番号： 112〜122のいずれかに記載のアミノ酸配列 において 1または複数のアミノ酸が置換、欠失、付加、および Zまたは挿入されたアミ ノ酸配列からなるタンパク質をコードする DNAが含まれる。  [0079] The DNA of the present invention includes an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence of any one of SEQ ID NOs: 112 to 122. DNA encoding a protein consisting of
[0080] 上記の DNAを調製するために、当業者によく知られた方法としては、例えば、 DNA に对し、 site-directed mutagenesis法 (Kramer, W. & Fritz, HJ" Methods Enzymol, 1 987, 154, 350.)により変異を導入する方法が挙げられる。  [0080] In order to prepare the above DNA, methods well known to those skilled in the art include, for example, a site-directed mutagenesis method (Kramer, W. & Fritz, HJ "Methods Enzymol, 1 987 , 154, 350.).
[0081] タンパク質におけるアミノ酸の改変は、通常、全アミノ酸の 50アミノ酸以内であり、好 ましくは 30アミノ酸以内であり、さらに好ましくは 10アミノ酸以内であり、さらに好ましく は 3アミノ酸以内である。アミノ酸の改変は、例えば、変異や置換であれば「Transform er Site— directed Mutagenesis Kit」や「ExSite PCR— Based ¾ite— directed Mutagenesis KitJ (Clontech社製）を用いて行うことが可能であり、また、欠失であれば「Quantum le ap Nested Deletion KitJ (Clontech社製）などを用いて行うことが可能である。  [0081] Modification of amino acids in a protein is usually within 50 amino acids of all amino acids, preferably within 30 amino acids, more preferably within 10 amino acids, and even more preferably within 3 amino acids. The amino acid modification can be performed using, for example, “Transformer Site—directed Mutagenesis Kit” or “ExSite PCR—Based ¾ite—directed Mutagenesis KitJ (Clontech), for mutation or substitution, Deletion can be performed using “Quantum le ap Nested Deletion Kit J (Clontech)”.
[0082] また、塩基配列が変異していても、その変異がタンパク質中のアミノ酸の変異を伴 わない場合 (縮重変異）があり、このような縮重変異 DNAも本発明に含まれる。 [0083] 本発明の DNAは、本発明のタンパク質をコードし得るものであれば特に制限はなく 、ゲノム DNA、 cDNA、化学合成 DNAなどが含まれる。ゲノム DNAは、例えば、文献（R ogers and Bendich, Plant Mol. Biol, 1985, 5, 69.)記載の方法に従って調製したゲノ ム DNAを铸型として、本発明の DNAの塩基配列（例えば、配列番号： 101〜111のい ずれかに記載の塩基配列）を基に作製したプライマーを用いて PCR(Saiki et al. Scie nce, 1988, 239, 487.)を行うことにより調製することが可能である。また、 cDNAであれ ί 、常法 (Maniatis et al. Molecular Cloningし old Spring haroor Laboratry Press)に より植物力 mRNAを調製し、逆転写反応を行い、上記と同様のプライマーを用いて P CRを行うことにより調製することが可能である。また、ゲノム DNAや cDNAは、常法によ りゲノム DNAライブラリーまたは cDNAライブラリーを作製し、このライブラリーに対し、 例えば本発明の DNAの塩基配列（例えば、配列番号： 101〜111のいずれかに記載 の塩基配列）を基に合成したプローブを用いてスクリーニングすることによつても調製 することが可能である。なお、得られた DNAの塩基配列は、例えば「シークェンサ一 M odel373j (ABI社製)を利用することにより容易に決定することが可能である。 [0082] Even if the base sequence is mutated, there is a case where the mutation does not involve a mutation of an amino acid in the protein (degenerate mutation), and such degenerate mutated DNA is also included in the present invention. [0083] The DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, cDNA, chemically synthesized DNA, and the like. Genomic DNA is, for example, a genomic DNA prepared according to the method described in the literature (Rogers and Bendich, Plant Mol. Biol, 1985, 5, 69.) as a saddle type, and the base sequence of the DNA of the present invention (for example, sequence It can be prepared by performing PCR (Saiki et al. Science, 1988, 239, 487.) using a primer prepared on the basis of the nucleotide sequence described in any of No. 101 to 111). is there. If cDNA is used, plant power mRNA is prepared by a conventional method (Maniatis et al. Molecular Cloning and old Spring haroor Laboratry Press), reverse transcription is performed, and PCR is performed using the same primers as above. Can be prepared. For genomic DNA or cDNA, a genomic DNA library or cDNA library is prepared by a conventional method. For example, the nucleotide sequence of the DNA of the present invention (for example, any one of SEQ ID NOs: 101 to 111) is prepared. It is also possible to prepare by screening using a probe synthesized based on the base sequence described above. The base sequence of the obtained DNA can be easily determined by using, for example, “Sequencer Model 373j (manufactured by ABI)”.
[0084] 本発明において、植物の転写因子をコードする DNAには、後述の「ユーカリ属カマ ルドレンシスの榭幹基部および榭幹中央部にお 、て、師部側と比較して木部側で発 現レベルが上昇している DNA」、「ユーカリ属カマルドレンシスの榭幹中央部において 、師部側と比較して木部側で発現レベルが上昇し、榭幹基部において、師部側と木 部側で発現レベルが同等な DNA」、「ユーカリ属カマルドレンシスおよびユーカリ属グ ロブルスの榭幹にお 、て、師部側と比較して木部側で発現レベルが上昇して 、る DN A」、「ユーカリ属カマルドレンシスの榭幹において、師部側と比較して木部側で発現 レベルが上昇し、ユーカリ属グロブルスの榭幹において、師部側と比較して木部側で 発現レベルが減少している DNA」、「ユーカリ属カマルドレンシスの榭幹において、師 部側と比較して木部側で発現レベルが上昇し、ユーカリ属グロブルスの樹幹にぉ ヽ て、師部側と木部側で発現レベルが同等な DNA」が含まれる。  [0084] In the present invention, the DNA encoding the plant transcription factor is described below in the xylem side compared to the phloem side in the trunk base and middle trunk of Eucalyptus genus Camaldrensis. The expression level rises in DNA ”,“ In the central trunk of Eucalyptus genus Camaldrensis, the expression level increases on the xylem side compared to the phloem side, and in the trunk base, The expression level on the xylem side is higher than that on the phloem side in the trunks of eucalyptus chamaldrensis and eucalyptus globulus. DN A ”,“ In the trunk of Eucalyptus genus Camaldrensis, the expression level increased on the xylem side compared to the phloem side, and in the trunk of the Eucalyptus globulus, the xylem side compared to the phloem side. DNA whose expression level is reduced by In the trunk of Sith, the expression level increased on the xylem side compared to the phloem side, and the expression level on the phloem globules trunk is equivalent to that on the phloem globules. It is.
[0085] 榭幹において、師部と木部では、木部側でのみ木繊維形成が行われることが知ら れている。よって、「師部側と比較して木部側で発現レベルが上昇または減少してい る DNA」は、「木繊維形成部位で発現レベルが上昇または減少して ヽる DNA」と表現 することもできる。また、榭幹基部の木部と榭幹中央部の木部では、榭幹基部の木部 と比較して榭幹中央部の木部の方が、木繊維形成 (性質)のうち木繊維長は長ぐ直 径は太ぐ細胞壁は厚ぐフィブリル傾角は緩ぐ木繊維率と比重は高くなる（成熟材 である）ことが知られている。また、榭幹中央部の木部と比較して榭幹基部の木部の 方力 木繊維形成のうち木繊維長は短ぐ直径は細ぐ細胞壁は薄ぐフィブリル傾角 は急で、木繊維率と比重は低くなる（未熟材である）ことが知られている。よって、「ュ 一カリの榭幹基部にぉ 、て、師部側と比較して木部側で発現レベルが上昇または減 少している DNA」は、「未熟材形成部位で発現レベルが上昇または減少している DNA 」と表現することもできる。また、「ユーカリの榭幹中央部において、師部側と比較して 木部側で発現レベルが上昇または減少している DNA」は、「成熟材形成部位で発現 レベルが上昇または減少して ヽる DNA」と表現することもできる。 [0085] In trunks, it is known that phloem and xylem form wood fiber only on the xylem side. Therefore, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side” is expressed as “DNA whose expression level increases or decreases at the wood fiber formation site”. You can also In addition, in the timber of the trunk base and the timber of the trunk trunk, the timber of the trunk trunk is more of the fiber length (characteristic) than the timber of the trunk trunk. It is known that the long fiber diameter is thick, the cell wall is thick, the fibril inclination is loose, and the wood fiber ratio and specific gravity are high (mature material). Also, compared with the xylem in the middle of the trunk, the xylem of the trunk of the trunk base Among the wood fiber formation, the wood fiber length is shorter, the diameter is thinner, the cell wall is thinner, the fibril inclination is steep, and the wood fiber rate It is known that the specific gravity is low (it is an immature material). Therefore, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side on the trunk base of a single pot” is expressed or increased in the immature wood formation site. It can also be expressed as “decreasing DNA”. In addition, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side in the central part of the eucalyptus trunk” is expressed in “ It can also be expressed as “DNA”.
[0086] さらに、カマルドレンシス榭幹の木部とグロブルス榭幹の木部では、カマルドレンシ ス榭幹の木部よりグロブルス榭幹の木部の方力 木繊維形成 (パルプ品質）のうち木 繊維長は長ぐ直径は太ぐ細胞壁は厚ぐフィブリル傾角は緩ぐ木繊維率と比重は 高くなることが知られている。また、グロブルス榭幹の木部よりカマルドレンシス榭幹の 木部の方が、木繊維長は短ぐ直径は細ぐ細胞壁は薄ぐフィブリル傾角は急で、 木繊維率と比重は低くなることが知られている。よって、「ユーカリ属カマルドレンシス の榭幹にぉ 、て、師部側と比較して木部側で発現レベルが上昇または減少して 、る DNA」は、「木繊維長は短ぐ直径は細ぐ細胞壁は薄ぐフィブリル傾角は急で、木 繊維率と比重は低 ヽ、と!ヽぅ低品質なパルプ特性を有する木繊維形成部位で発現レ ベルが上昇または減少している DNA」と表現することもできる。また、「ユーカリ属グロ プルスの樹幹にぉ 、て、師部側と比較して木部側で発現レベルが上昇または減少し ている DNA」は、「木繊維長は長ぐ直径は太ぐ細胞壁は厚ぐフィブリル傾角は緩く 、木繊維率と比重は高い、という高品質なパルプ特性を有する木繊維を形成する部 位で発現レベルが上昇または減少して ヽる DNA」と表現することもできる。  [0086] Further, in the xylem of the Camaldrensis trunk and the grouse of the Globulus trunk, the direction of the xylem of the Grobles trunk from the xylem of the Camaldrensis trunk is the fiber of the wood fiber formation (pulp quality) It is known that the length and diameter are thick and the cell wall is thick and the fibril inclination is loose and the wood fiber ratio and specific gravity are high. Also, the xylem of the Camaldrensis trunk is shorter than the globules trunk, but the diameter of the fiber is shorter, the cell wall is thinner, the fibril inclination is steeper, and the wood fiber ratio and specific gravity are lower. It has been known. Therefore, “DNA that has an increased or decreased expression level on the xylem side compared to the phloem side, compared to the trunk of the Eucalyptus genus Camaldrensis” `` The thin cell walls are thin, the fibril inclination is steep, the wood fiber rate and specific gravity are low, and the expression level is increased or decreased at the wood fiber formation site with low quality pulp characteristics. '' It can also be expressed. In addition, “DNA with an increased or decreased expression level on the xylem side compared to the phloem side on the trunk of Eucalyptus globulus” means that “wood fiber length is longer and diameter is thicker than the cell wall. It can be expressed as `` DNA whose expression level is increased or decreased in the part that forms wood fiber with high quality pulp characteristics that thick fibril inclination is gentle, wood fiber rate and specific gravity are high '' .
[0087] 本発明にお 、て、師部側とは***組織である維管束形成層より外側を意味し、好 ましくは師部側の榭皮形成部位を意味する。ここで、榭皮形成部位とは師細胞、師管 、柔細胞、じん皮細胞等へ分ィ匕中またはその分ィ匕能力を有する部位である。また、本 発明において、木部側とは維管束形成層より内側を意味し、好ましくは木部側の木 繊維形成部位を意味する。ここで、木繊維形成部位とは木繊維細胞、仮導管、導管 、柔細胞等へ分ィ匕中またはその分ィ匕能力を有する部位である。 In the present invention, the phloem side means the outside of the vascularization layer that is a meristem, and preferably the scab formation site on the phloem side. Here, the scab formation site is a site that is in the middle of or is capable of splitting into phloem cells, phloem tubules, parenchyma cells, and pelvic cells. Also book In the present invention, the xylem side means the inner side of the vascular bundle forming layer, preferably the xylem side wood fiber forming site. Here, the term “wood fiber forming site” refers to a site that is being divided into wood fiber cells, temporary conduits, conduits, parenchymal cells, etc., or that has the ability to separate them.
[0088] 本発明において、榭幹基部とは、一般的にいわれる胸高部未満を指し、例えば、ュ 一カリの 5年生の植物体の場合、地上高約 lm未満を指す。また、榭幹中央部とは、 一般的に榭高の中間部付近を指し、例えば、榭高 10m以上のユーカリの 5年生の植 物体の場合、地上高約 5m付近を指す。 [0088] In the present invention, the trunk base means a generally called breast height, for example, in the case of a 5- year-old plant of Eucalyptus, it indicates a height of less than about lm above the ground. In addition, the central part of the trunk generally refers to the middle part of the culm height, for example, in the case of a eucalyptus fifth-year plant that has a culm height of 10 m or more, it refers to a height of about 5 m above the ground.
[0089] 本発明の DNAまたはその部分 DNAは、植物の榭幹木部 (好ましくは榭幹基部また は榭幹中央部の木部）の生長状態の検査用マーカーとして利用できる。すなわち、 上記 DNAまたはその部分 DNAを少なくとも 1つ利用することで、植物の榭幹木部 (好 ましくは榭幹基部または榭幹中央部の木部)の生長状態を検査することができる。  [0089] The DNA of the present invention or a partial DNA thereof can be used as a marker for examining the growth state of a trunk of a plant (preferably, a xylem of a trunk trunk or a middle part of a trunk). That is, by using at least one of the above-mentioned DNAs or partial DNAs thereof, the growth state of the trunk trunk of the plant (preferably the trunk of the trunk trunk or the central part of the trunk) can be examined.
[0090] より具体的には、以下の（1)または（2)の場合に、植物の榭幹木部が、木繊維形成 が盛んな木部であると判定される。さらに、（2)の場合に、植物の榭幹木部が、成熟 材が形成されて 、る木部、または将来的に成熟材が形成される木部であると判定さ れる。ここで、成熟材とは、成熟した形成層組織から分化して形成される木繊維を意 味し、同様な条件で採取された榭幹基部の木部と比較して、木繊維長は長ぐ直径 は太ぐ細胞壁は厚ぐフィブリル傾角は緩ぐ木繊維率と比重は高いという特徴を有 する。また、未熟材とは、未熟な形成層組織から分化'形成される木繊維を意味し、 同様な条件で採取された榭幹中央部の木部と比較して、木繊維長は短ぐ直径は細 ぐ細胞壁は薄ぐフィブリル傾角は急で、木繊維率と比重は低いという特徴を有する  More specifically, in the case of the following (1) or (2), it is determined that the trunk trunk portion of the plant is a xylem portion in which wood fiber formation is active. Further, in the case of (2), it is determined that the trunk trunk portion of the plant is a xylem portion where mature wood is formed, or a xylem where mature wood is formed in the future. Here, mature wood means wood fibers that are formed by differentiation from a mature formation layer structure, and the wood fiber length is longer than that of the trunk of the trunk base collected under the same conditions. The cell wall is thick, the cell wall is thick, the fibril inclination is loose, and the wood fiber ratio and specific gravity are high. In addition, immature wood means wood fibers that are differentiated and formed from an immature formed layer structure, and the length of the wood fiber is shorter than that of the wood part of the central trunk collected under the same conditions. The cell wall is thin and the fibril inclination is steep, and the wood fiber rate and specific gravity are low.
[0091] 本発明の検査に用いられる植物は、好ましくはユーカリ、より好ましくはユーカリ属カ マルドレンシス種またはユーカリ属グロブルス種である。 [0091] The plant used in the test of the present invention is preferably Eucalyptus, more preferably Eucalyptus Camaldlensis or Eucalyptus Globulus.
[0092] また、本発明の検査方法を利用することで材質の量的 ·質的な事前予測が可能とな る。また、植林事業において、生長状態をもとに適切な施業 (除草'間伐等)を適切な 時期に施すことができる。例えば、本発明における榭幹基部の師部と木部での発現 ノターンと本発明の検査方法で得られた発現パターンに相関があれば (好ましくはピ ァソンの相関係数 0.5以上、より好ましくは 0.7以上、以下同じ）、被検榭幹木部に、質 的には未熟な繊維 (未熟材:短繊維、直径は細ぐ細胞壁は薄ぐフィブリル傾角は 急で、低木繊維率と低比重）が形成されている、もしくは将来的に形成される状態で あり、木繊維量 (容積重)も少な 、もしくは将来的に少なくなる（平均的な容積重 450 〜500kg/m3以下）という予測ができる。一方、本発明における榭幹中央部の師部と木 部での発現パターンと本発明の検査方法で得られた発現パターンに相関があれば、 被検榭幹木部に、質的には成熟した繊維 (成熟材:長繊維、直径は太ぐ細胞壁は 厚ぐフィブリル傾角は緩ぐ高木繊維率と高比重)が形成されている、もしくは将来 的に形成される状態であり、木繊維量 (容積重)も多いもしくは将来的に多くなる（平 均的な容積重 450〜500kg/m3以上）という予測ができる。さらに、上記のような材質予 測により、植物の生長状態の善し悪しが明ら力となるため、悪いと判断されれば、貧 栄養地では施肥を行ったり、雑草が茂っていれば除草作業を早期に行ったり、植栽 密度が高ければ間伐を行ったり、といった施業を迅速かつ適切に行うことが可能にな る。現在、前述のような材質予測や施業方針の決定は、多分に現場の経験や勘、も しくは事後の化学分析等に頼っており、本願で示すような事前に判別できる科学的 指標は無い。 [0092] Further, by using the inspection method of the present invention, quantitative and qualitative advance prediction of a material becomes possible. In afforestation projects, appropriate operations (weeding, thinning, etc.) can be carried out at appropriate times based on the growth conditions. For example, if there is a correlation between the expression pattern in the phloem and xylem of the trunk base in the present invention and the expression pattern obtained by the test method of the present invention (preferably the correlation coefficient of Pearson is 0.5 or more, more preferably 0.7 or higher, the same shall apply hereinafter) In fact, immature fibers (immature wood: short fibers, thin diameter cell walls are thin, fibril inclination is steep, shrub fiber rate and low specific gravity) are formed or are in a state of being formed in the future Therefore, it can be predicted that the amount of wood fibers (volume weight) will be small or will decrease in the future (average volume weight 450 to 500 kg / m 3 or less). On the other hand, if there is a correlation between the expression pattern in the phloem and xylem of the center of the trunk in the present invention and the expression pattern obtained by the test method of the present invention, the maturity qualitatively in the subject trunk Fibers (mature material: long fibers, thicker cell walls are thicker, fibril inclination is thicker and higher wood fiber ratio and high specific gravity) are formed, or are formed in the future, and the amount of wood fibers ( It can be predicted that there will be a large (increase in specific gravity) or increase in the future (an average specific gravity of 450 to 500 kg / m 3 or more). Furthermore, the material quality prediction as described above will clearly reveal the quality of the plant's growth, so if it is judged to be bad, fertilizer is applied in an oligotrophic area or weeding work is carried out if weeds are thick. It is possible to conduct operations quickly and appropriately, such as early implementation or thinning if the planting density is high. Currently, material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance as shown in this application. .
(1)植物の榭幹 (好ましくは榭幹基部または榭幹中央部）の師部側および木部側に おいて、「ユーカリ属カマルドレンシスの榭幹基部および榭幹中央部において、師部 側と比較して木部側で発現レベルが上昇している DNA」のうち少なくとも 1つ (好ましく は複数、より好ましくは全て。以下同じ)の DNAの発現レベルを検出し、師部側および 木部側における該 DNAの発現レベルを比較した結果、植物の榭幹における該 DNA の発現レベル力 師部側と比較して木部側で上昇して 、る場合  (1) On the phloem side and the xylem side of the trunk of the plant (preferably the trunk trunk or the middle of the trunk), “the phloem in the trunk trunk and the middle trunk of the Eucalyptus genus Camaldrensis” The expression level of at least one (preferably a plurality, more preferably all, the same below) of DNA whose expression level is increased on the xylem side relative to the xylem side is detected. As a result of comparing the expression level of the DNA on the xylem side, the expression level of the DNA on the trunk of the plant is higher on the xylem side than on the phloem side
本発明にお 、て、「ユーカリ属カマルドレンシスの榭幹基部および榭幹中央部にお いて、師部側と比較して木部側で発現レベルが上昇している DNA」としては、以下の (a)〜（e)の!、ずれかに記載の DNAが挙げられる。  In the present invention, “DNA having an increased expression level on the xylem side compared to the phloem side in the trunk base and middle trunk of Eucalyptus genus Camaldrensis” Of (a) to (e), or any of the DNAs described in any of them.
(a)配列番号： 113〜 117の 、ずれかに記載のアミノ酸配列力 なるタンパク質をコ ードする DNA  (a) DNA encoding a protein having the amino acid sequence ability described in any one of SEQ ID NOS: 113 to 117
(b)配列番号： 102〜106のいずれかに記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence according to any one of SEQ ID NOs: 102 to 106
(c)配列番号： 113〜 117の 、ずれかに記載のアミノ酸配列力もなるタンパク質と 50 %以上の相同性を有するタンパク質をコードする DNA (c) SEQ ID NOs: 113 to 117 DNA encoding a protein with a homology of at least%
(d)配列番号： 102〜 106のいずれかに記載の塩基配列からなる DNAとストリンジェ ントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 102 to 106
(e)配列番号： 113〜 117の 、ずれかに記載のアミノ酸配列にお 、て 1または複数の アミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配列力 なるタンパ ク質をコードする DNA  (e) SEQ ID NO: 113-117, which encodes a protein having an amino acid sequence ability in which one or more amino acids are substituted, deleted, added, and Z or inserted in any one of the amino acid sequences described in any one of DNA
「配列番号： 102〜106のいずれかに記載の塩基配列からなる DNA」としては、配 列番号： 102、 105または 106のいずれかに記載の塩基配列力もなる DNAが好ましく 、配列番号： 105または 106に記載の塩基配列力もなる DNAがより好ましいが、これら に限定されるものではない。  As the “DNA consisting of the base sequence described in any one of SEQ ID NOs: 102 to 106”, DNA having the base sequence ability described in any one of SEQ ID NOs: 102, 105 or 106 is preferable, and SEQ ID NO: 105 or DNA having a nucleotide sequence ability described in 106 is more preferable, but is not limited thereto.
(2)植物の榭幹 (好ましくは榭幹中央部）の師部側および木部側にお 、て、「ユー カリ属カマルドレンシスの榭幹中央部にぉ 、て、師部側と比較して木部側で発現レ ベルが上昇し、榭幹基部において、師部側と木部側で発現レベルが同等な DNA」の うち少なくとも 1つの DNAの発現レベルを検出し、師部側および木部側における該 D NAの発現レベルを比較した結果、植物の榭幹における該 DNAの発現レベル力 師 部側と比較して木部側で上昇して!/ヽる場合  (2) On the phloem side and xylem side of the trunk of the plant (preferably in the middle of the trunk), compare with the phloem side in the middle of the trunk of Eucalyptus genus Camaldrensis. Then, the expression level rises on the xylem side, and at the trunk base, the expression level of at least one DNA of `` DNA with the same expression level on the phloem side and the xylem side '' is detected. When the expression level of the DNA on the xylem side is compared, the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side!
本発明において、「ユーカリ属カマルドレンシスの榭幹中央部において、師部側と 比較して木部側で発現レベルが上昇し、榭幹基部において、師部側と木部側で発現 レベルが同等な DNA」としては、以下の（a)〜（e)の!、ずれかに記載の DNAが挙げら れる。  In the present invention, the expression level is increased on the xylem side compared to the phloem side in the central trunk of Eucalyptus genus Camaldrensis, and the expression level is increased on the phloem side and xylem side in the trunk base. Examples of “equivalent DNA” include the DNAs described in the following (a) to (e)!
(a)配列番号： 112、 118または 120〜122のいずれかに記載のアミノ酸配列からな るタンパク質をコードする DNA  (a) DNA encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 112, 118 or 120-122
(b)配列番号： 101、 107または 109〜111のいずれかに記載の塩基配列を含む DN A  (b) SEQ ID NO: 101, 107 or DN A containing the base sequence described in 109 to 111
(c)配列番号： 112、 118または 120〜122のいずれかに記載のアミノ酸配列からな るタンパク質と 50%以上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with a protein consisting of the amino acid sequence of SEQ ID NO: 112, 118 or 120-122
(d)配列番号： 101、 107または 109〜111のいずれかに記載の塩基配列からなる D NAとストリンジェントな条件下でハイブリダィズする DNA (e)配列番号： 112、 118または 120〜 122の!、ずれかに記載のアミノ酸配列にお！/ヽ て 1または複数のアミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配 列からなるタンパク質をコードする DNA (d) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 101, 107 or 109 to 111 (e) SEQ ID NO: 112, 118 or 120-122! DNA that encodes a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted.
「配列番号： 101、 107または 109〜111のいずれかに記載の塩基配列からなる D NA」としては、配列番号： 107または 109〜111のいずれかに記載の塩基配列から なる DNAが好ましぐ配列番号： 107または 109に記載の塩基配列からなる DNAがよ り好まし 、が、これらに限定されるものではな！/、。  As the “DNA consisting of the nucleotide sequence of SEQ ID NO: 101, 107 or 109 to 111”, DNA consisting of the nucleotide sequence of SEQ ID NO: 107 or 109 to 111 is preferred. DNA having the nucleotide sequence set forth in SEQ ID NO: 107 or 109 is more preferred, but is not limited to these! /.
さらに、本発明においては、異なる 2つの榭幹木部の生長状態 (成熟度)を検査す る方法を提供する。すなわち、異なる 2つの榭幹木部うち、どちらが成熟しているのか 、または未熟であるのかを検査する方法を提供する。本発明において、「異なる 2つ の榭幹木部」には、異なる 2つの植物における榭幹木部、および、 1つの植物におけ る異なる 2つの榭幹木部が含まれる。上記方法は、複数の榭幹木部から、成熟した、 または未熟な榭幹木部を選別することにも使用できる。  Furthermore, the present invention provides a method for examining the growth state (maturity) of two different trunks. In other words, it provides a method for examining which of two different trunks are mature or immature. In the present invention, “two different trunk trunks” include two trunk trunks in two different plants and two different trunk trunks in one plant. The above method can also be used to select mature or immature trunk trunks from a plurality of trunk trunks.
より具体的には、以下の（3)の場合に、異なる 2つの榭幹木部のうち、片方の榭幹 木部 (以下、榭幹木部 Aと称す)と比較して他方の榭幹木部 (以下、榭幹木部 Bと称す )は、成熟している木部であり、榭幹木部 Bと比較して榭幹木部 Aは、未熟な木部であ ると判定される。従って、以下の（3)は、例えば育種現場において、エリート木やブラ スツリーと呼ばれる優良系統を選抜するときの指標になる。現在、エリート木等の選抜 は、榭高ゃ直径などの外見上の数値や伐採後の材質分析値を指標に行われており 、上記指標で選抜する技術 (概念)は無い。  More specifically, in the case of (3) below, of the two different trunk trunks, the other trunk trunk is compared with one trunk trunk (hereinafter referred to as trunk trunk A). The xylem (hereinafter referred to as “trunk trunk B”) is a mature xylem, and compared to “trunk trunk timber B”, the trunk xylem A is determined to be an immature xylem. The Therefore, the following (3) is an index for selecting excellent lines called elite trees and brass trees, for example, in breeding sites. At present, selection of elite trees and the like is performed using an apparent numerical value such as the diameter of a spider and a material analysis value after cutting, and there is no technique (concept) for selecting with the above index.
(3)異なる 2つの榭幹木部において、「ユーカリの榭幹中央部の木部側と比較して 榭幹基部の木部側で発現レベルが減少している DNA」のうち少なくとも 1つの DNAの 発現レベルを検出し、異なる 2つの榭幹木部における該 DNAの発現レベルを比較し た結果、該 DNAの発現レベル力 榭幹木部 Bと比較して榭幹木部 Aで減少している (3) At least one DNA in two different trunk trunks, `` DNA whose expression level is decreased on the xylem side of the trunk base compared to the xylem side of the middle part of the eucalyptus trunk '' As a result of detecting the expression level of the DNA and comparing the expression level of the DNA in two different trunk trunks, the expression level of the DNA decreased in the trunk trunk A compared to the trunk trunk B. Have
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本発明において、「ユーカリの榭幹中央部の木部側と比較して榭幹基部の木部側 で発現レベルが減少して 、る DNA」としては、以下の（a)〜（e)の!、ずれかに記載の DNAが挙げられる。 (a)配列番号： 113または 114に記載のアミノ酸配列力もなるタンパク質をコードする DNA In the present invention, “DNA whose expression level is reduced on the xylem side of the trunk base as compared with the xylem side of the eucalyptus trunk center” is the following (a) to (e): !, The DNA described in somewhere. (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 113 or 114
(b)配列番号： 102または 103に記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 102 or 103
(c)配列番号： 113または 114の 、ずれかに記載のアミノ酸配列力もなるタンパク質と 50%以上の相同性を有するタンパク質をコードする DNA  (c) a DNA encoding a protein having 50% or more homology with a protein of SEQ ID NO: 113 or 114 that also has an amino acid sequence ability according to any one of
(d)配列番号： 102または 103のいずれかに記載の塩基配列からなる DNAとストリン ジェントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 102 or 103
(e)配列番号： 113または 114の 、ずれかに記載のアミノ酸配列にお 、て 1または複 数のアミノ酸が置換、欠失、付加、および/または挿入されたアミノ酸配列からなるタ ンパク質をコードする DNA  (e) a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into any one of the amino acid sequences of SEQ ID NO: 113 or 114 DNA to encode
「配列番号： 102または 103に記載の塩基配列力もなる DNA」としては、配列番号： 102に記載の塩基配列力もなる DNAが好ましいが、これらに限定されるものではない また本発明においては、以下の (4)〜（7)の場合に、植物の榭幹木部が、木繊維 形成が盛んな木部であると判定される。さらに、（5)または（6)の場合に、植物の榭幹 木部が、同様な条件で採取されたグロプルスの樹幹の木部と比較して、木繊維長は 短ぐ直径は細ぐ細胞壁は薄ぐフィブリル傾角は急で、木繊維率と比重は低い、と V、うパルプ特性を有する木繊維が形成されて!ヽる木部、または将来的に該パルプ特 性を有する木繊維が形成される木部であると判定される。また、（7)の場合に、植物 の榭幹木部が、同様な条件で採取されたカマルドレンシス榭幹の木部と比較して、木 繊維長は長ぐ直径は太ぐ細胞壁は厚ぐフィブリル傾角は緩ぐ木繊維率と比重は 高い、という優れたパルプ特性を有する木繊維が形成されている木部、または将来 的に該パルプ特性を有する木繊維が形成される木部であると判定される。  The “DNA having the nucleotide sequence ability described in SEQ ID NO: 102 or 103” is preferably a DNA having the nucleotide sequence ability described in SEQ ID NO: 102, but is not limited thereto. In the case of (4) to (7), it is determined that the trunk stem portion of the plant is a xylem portion in which wood fiber formation is thriving. Furthermore, in the case of (5) or (6), the length of the fiber of the trunk of the plant is shorter and the diameter of the cell wall is narrower than that of the grops trunk of the groprus collected under the same conditions. The fibril inclination angle is steep, the wood fiber ratio and specific gravity are low, and V, a wood fiber having a pulp characteristic is formed! It is determined that the xylem is formed. In addition, in the case of (7), the length of the tree fiber is longer and the cell wall is thicker than the length of the stem of the trunk of the plant compared to the xylem of the Camaldrensis trunk collected under the same conditions. It is a xylem where wood fibers with excellent pulp properties are formed, such as the ratio of loose fibrils is high and the specific gravity is high, or the xylem where wood fibers with the pulp properties are formed in the future It is determined.
また、本発明の検査方法を利用することで材質の量的 ·質的な事前予測が可能とな る。また、植林事業において、生長状態をもとに適切な施業 (除草'間伐等)を適切な 時期に施すことができる。例えば、本発明におけるユーカリ属カマルドレンシス榭幹 の師部と木部での発現パターンと本発明の検査方法で得られた発現パターンに相 関があれば (好ましくはピアソンの相関係数 0.5以上、より好ましくは 0.7以上、以下同 じ)、被検榭幹木部に、質的には低レベルな木繊維 (低品質パルプ:短繊維、直径は 細ぐ細胞壁は薄ぐフィブリル傾角は急で、低木繊維率と低比重）が形成されている 、もしくは将来的に形成される状態であり、木繊維量 (容積重)も少ないもしくは将来 的に少なくなる（平均的な容積重 450〜500kg/m3以下）という予測ができる。一方、本 発明におけるユーカリ属グロブルス榭幹の師部と木部での発現パターンと本発明の 検査方法で得られた発現パターンに相関があれば、被検榭幹木部に、質的には優 れた繊維 (高品質パルプ:長繊維、直径は太ぐ細胞壁は厚ぐフィブリル傾角は緩く 、高木繊維率と高比重）が形成されている、もしくは将来的に形成される状態であり、 木繊維量 (容積重)も多 、もしくは将来的に多くなる（平均的な容積重 450〜500kg/m 3以上）という予測ができる。さらに、上記のような材質予測により、植物の生長状態の 善し悪しが明ら力となるため、悪いと判断されれば、貧栄養地では施肥を行ったり、 雑草が茂っていれば除草作業を早期に行ったり、植栽密度が高ければ間伐を行つ たり、といった施業を迅速かつ適切に行うことが可能になる。現在、前述のような材質 予測や施業方針の決定は、多分に現場の経験や勘、もしくは事後の化学分析等に 頼っており、本願で示すような事前に判別できる科学的指標は無い。 In addition, by using the inspection method of the present invention, quantitative and qualitative advance prediction of materials can be performed. In afforestation projects, appropriate operations (weeding, thinning, etc.) can be carried out at appropriate times based on the growth conditions. For example, if there is a correlation between the expression pattern in the phloem and xylem of Eucalyptus genus Camaldrensis trunk in the present invention and the expression pattern obtained by the test method of the present invention (preferably Pearson's correlation coefficient of 0.5 or more , More preferably 0.7 or more, the same below Ji), low-quality wood fibers (low quality pulp: short fibers, thin cell walls are thin, fibril inclination is steep, shrub fiber ratio and low specific gravity) It can be predicted that it will be formed or will be formed in the future, and the amount of wood fiber (volume weight) will be small or will decrease in the future (average weight of 450 to 500 kg / m 3 or less). On the other hand, if there is a correlation between the expression pattern in the phloem and xylem of the Eucalyptus globulus trunk in the present invention and the expression pattern obtained by the test method of the present invention, Excellent fibers (high quality pulp: long fibers, thicker cell walls are thicker, fibril inclination is gentle, high wood fiber ratio and high specific gravity) are formed, or are formed in the future. It can be predicted that the amount of fiber (volume weight) will be large or will increase in the future (average weight of 450 to 500 kg / m 3 or more). In addition, the material quality prediction as described above makes it clear that the state of plant growth is good or bad, so if it is judged to be bad, fertilization is performed in an oligotrophic area, or if weeds are overgrown, early weeding work is performed. If the planting density is high, thinning can be performed quickly and appropriately. At present, material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance.
(4)植物の榭幹 (好ましくは榭幹基部または榭幹中央部）の師部側および木部側に おいて、「ユーカリ属カマルドレンシスおよびユーカリ属グロブルスの榭幹において、 師部側と比較して木部側で発現レベルが上昇している DNA」のうち少なくとも 1つの D NAの発現レベルを検出し、師部側および木部側における該 DNAの発現レベルを比 較した結果、植物の榭幹における該 DNAの発現レベル力 師部側と比較して木部側 で上昇している場合  (4) On the phloem side and xylem side of the trunk of the plant (preferably the trunk base or the middle of the trunk), “In the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules, As a result of detecting the expression level of at least one DNA out of `` DNA whose expression level is increased on the xylem side compared with the other '', the expression level of the DNA on the phloem side and the xylem side was compared. The level of expression of the DNA in the trunk of the tree is higher on the xylem side than on the phloem side
本発明にお 、て、「ユーカリ属カマルドレンシスおよびユーカリ属グロブルスの榭幹 において、師部側と比較して木部側で発現レベルが上昇している DNA」としては、以 下の（a)〜（e)の!、ずれかに記載の DNAが挙げられる。  In the present invention, “DNA having an increased expression level on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules” includes the following (a ) To (e)!
(a)配列番号： 114または 116に記載のアミノ酸配列力もなるタンパク質をコードする DNA  (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 114 or 116
(b)配列番号： 103または 105に記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 103 or 105
(c)配列番号： 114または 116に記載のアミノ酸配列力もなるタンパク質と 50%以上 の相同性を有するタンパク質をコードする DNA (c) 50% or more of the protein having the amino acid sequence ability described in SEQ ID NO: 114 or 116 DNA encoding a protein with homology of
(d)配列番号： 103または 105に記載の塩基配列からなる DNAとストリンジヱントな条 件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 103 or 105
(e)配列番号： 114または 116に記載のアミノ酸配列にお 、て 1または複数のアミノ酸 が置換、欠失、付加、および/または挿入されたアミノ酸配列力 なるタンパク質をコ ードする DNA  (e) DNA encoding a protein having amino acid sequence ability in which one or more amino acids are substituted, deleted, added and / or inserted into the amino acid sequence set forth in SEQ ID NO: 114 or 116
「配列番号： 103または 105に記載の塩基配列力もなる DNA」としては、配列番号： 105に記載の塩基配列力 なる DNAが好ましいが、これらに限定されるものではない (5)植物の榭幹 (好ましくは榭幹基部または榭幹中央部）の師部側および木部側に おいて、「ユーカリ属カマルドレンシスの榭幹において、師部側と比較して木部側で 発現レベルが上昇し、ユーカリ属グロブルスの榭幹において、師部側と比較して木部 側で発現レベルが減少して 、る DNA」のうち少なくとも 1つの DNAの発現レベルを検 出し、師部側および木部側における該 DNAの発現レベルを比較した結果、植物の榭 幹における該 DNAの発現レベル力 師部側と比較して木部側で上昇して 、る場合 本発明において、「ユーカリ属カマルドレンシスの榭幹において、師部側と比較して 木部側で発現レベルが上昇し、ユーカリ属グロブルスの榭幹において、師部側と比 較して木部側で発現レベルが減少して!/ヽる DNA」としては、以下の（a)〜（e)の!ヽず れかに記載の DNAが挙げられる。  The “DNA having a nucleotide sequence ability described in SEQ ID NO: 103 or 105” is preferably a DNA having a nucleotide sequence ability described in SEQ ID NO: 105, but is not limited thereto. (5) Plant trunk On the phloem side and the xylem side of the trunk base (preferably the trunk base or the trunk trunk center), the expression level increased on the xylem side compared to the phloem side in the trunk of the Eucalyptus genus Camaldrensis However, in the trunk of Eucalyptus globules, the expression level is decreased on the xylem side compared to the phloem side, and the expression level of at least one DNA is detected. As a result of comparing the expression level of the DNA on the side, the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side. Compared to the phloem side, The expression level rises on the part side, and the expression level decreases on the xylem side compared to the phloem side in the trunk of Eucalyptus globules! The DNA described in (e)!
(a)配列番号： 115または 117に記載のアミノ酸配列力もなるタンパク質をコードする DNA  (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 115 or 117
(b)配列番号： 104または 106に記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 104 or 106
(c)配列番号： 115または 117に記載のアミノ酸配列力もなるタンパク質と 50%以上 の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in SEQ ID NO: 115 or 117
(d)配列番号： 104または 106に記載の塩基配列からなる DNAとストリンジェントな条 件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 104 or 106
(e)配列番号： 115または 117に記載のアミノ酸配列にお 、て 1または複数のアミノ酸 が置換、欠失、付加、および/または挿入されたアミノ酸配列力 なるタンパク質をコ ードする DNA (e) A protein having an amino acid sequence ability in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence set forth in SEQ ID NO: 115 or 117 DNA
[0098] 「配列番号： 104または 106に記載の塩基配列力もなる DNA」としては、配列番号： 106に記載の塩基配列力 なる DNAが好ましいが、これらに限定されるものではない  [0098] The DNA having the nucleotide sequence ability described in SEQ ID NO: 104 or 106 is preferably the DNA having the nucleotide sequence ability described in SEQ ID NO: 106, but is not limited thereto.
[0099] (6)植物の榭幹 (好ましくは榭幹基部または榭幹中央部）の師部側および木部側に おいて、「ユーカリ属カマルドレンシスの榭幹において、師部側と比較して木部側で 発現レベルが上昇し、ユーカリ属グロブルスの榭幹において、師部側と木部側で発 現レベルが同等な DNA」のうち少なくとも 1つの DNAの発現レベルを検出し、師部側 および木部側における該 DNAの発現レベルを比較した結果、植物の榭幹における 該 DNAの発現レベル力 師部側と比較して木部側で上昇して 、る場合 [0099] (6) On the phloem side and the xylem side of the trunk of the plant (preferably the trunk trunk or the middle of the trunk), compared with the phloem side in the trunk of Eucalyptus genus Camaldrensis As a result, the expression level rises on the xylem side, and the expression level of at least one of the DNAs with the same expression level on the phloem side and xylem side is detected in the trunk of the Eucalyptus globules. As a result of comparing the expression level of the DNA on the xylem side and the xylem side, the expression level of the DNA on the trunk of the plant is higher on the xylem side than on the phloem side
本発明において、「ユーカリ属カマルドレンシスの榭幹において、師部側と比較して 木部側で発現レベルが上昇し、ユーカリ属グロブルスの榭幹において、師部側と木 部側で発現レベルが同等な DNA」としては、以下の（a)〜（e)のいずれかに記載の D NAが挙げられる。  In the present invention, the expression level is increased on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis, and the expression level on the phloem side and xylem side in the trunk of the Eucalyptus globules Examples of the DNA having the same DNA include DNAs described in any of the following (a) to (e).
(a)配列番号： 113に記載のアミノ酸配列力もなるタンパク質をコードする DNA (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 113
(b)配列番号： 102に記載の塩基配列を含む DNA (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 102
(c)配列番号：113に記載のアミノ酸配列力もなるタンパク質と 50%以上の相同性を 有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in SEQ ID NO: 113
(d)配列番号： 102に記載の塩基配列からなる DNAとストリンジ ントな条件下でノ、ィ ブリダィズする DNA  (d) DNA that is hybridized under stringent conditions with DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 102
(e)配列番号： 113に記載のアミノ酸配列において 1または複数のアミノ酸が置換、欠 失、付加、および Zまたは挿入されたアミノ酸配列からなるタンパク質をコードする DN A  (e) DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence set forth in SEQ ID NO: 113
[0100] (7)植物の榭幹 (好ましくは榭幹中央部）の師部側および木部側において、上記「 ユーカリ属カマルドレンシスの樹幹にぉ 、て、師部側と比較して木部側で発現レベル が上昇し、ユーカリ属グロブルスの榭幹において、師部側と比較して木部側で発現レ ベルが減少している DNA」のうち少なくとも 1つの DNAの発現レベルを検出し、師部 側および木部側における該 DNAの発現レベルを比較した結果、植物の榭幹におけ る該 DNAの発現レベルが、師部側と比較して木部側で減少して ヽる場合 さらに、本発明においては、異なる 2つの植物 (好ましくはユーカリ）の榭幹木部の 生長状態 (パルプ品質)を検査する方法を提供する。すなわち、異なる 2つの植物の 榭幹木部うち、どちらが優れたパルプ品質なのか、または劣るのかを検査する方法を 提供する。この方法は、複数の植物の榭幹木部から、優れた、または劣った榭幹木 部を選別することにも使用できる。 [0100] (7) On the phloem side and the xylem side of the trunk of the plant (preferably in the middle of the trunk), the tree of the Eucalyptus genus Camaldrensis is compared to the phloem side. In the trunk of Eucalyptus globules, the expression level of at least one DNA is detected in the eucalyptus globulus trunk, the expression level of which is reduced on the xylem side compared to the phloem side. As a result of comparing the expression level of the DNA on the phloem side and xylem side, In addition, in the present invention, the growth state of the trunk stem part of two different plants (preferably eucalyptus) is compared with the phloem side. A method for inspecting pulp quality is provided. In other words, it provides a method for inspecting which of two different plant trunks is superior or inferior in pulp quality. This method can also be used to select good or inferior trunk trunks from multiple plant trunk trunks.
[0101] より具体的には、以下の（11)の場合に、異なる 2つの植物の榭幹木部のうち、片方 の植物（以下、植物 Aと称す)の榭幹木部と比較して他方の植物（以下、植物 Bと称す )の榭幹木部は、優れたパルプ品質を有する木部であり、榭幹木部 Bと比較して榭幹 木部 Aは、劣ったパルプ品質を有する木部であると判定される。従って、以下の（11) は、例えば育種現場において、エリート木やプラスツリーと呼ばれる優良系統を選抜 するときの指標になる。現在、エリート木等の選抜は、榭高ゃ直径などの外見上の数 値や伐採後の材質分析値を指標に行われており、上記指標で選抜する技術 (概念） は無い。 [0101] More specifically, in the case of (11) below, it is compared with the trunk trunk of one of the two different plants (hereinafter referred to as plant A). The trunk of the other plant (hereinafter referred to as plant B) is a xylem having an excellent pulp quality. Compared with the trunk xylem B, the trunk xylem A has an inferior pulp quality. It is determined that the xylem has. Therefore, the following (11) is an index when selecting excellent lines called elite trees and plus trees, for example, at breeding sites. At present, selection of elite trees, etc. is performed using the numerical values such as the diameter and the material analysis values after cutting, and there is no technology (concept) to select using the above indicators.
[0102] (8)異なる 2つの植物の榭幹木部 (好ましくは榭幹基部または榭幹中央部の木部） にお 、て、「ユーカリ属グロブルスの樹幹の木部側と比較してユーカリ属カマルドレン シスの榭幹の木部側で発現レベルが減少して 、る DNA」のうち少なくとも 1つの DNA の発現レベルを検出し、異なる 2つの植物の榭幹木部における該 DNAの発現レベル を比較した結果、該 DNAの発現レベル力 植物 Bと比較して植物 Aで減少している場 本発明にお 、て、 「ユーカリ属グロブルスの樹幹の木部側と比較してユーカリ属カ マルドレンシスの樹幹の木部側で発現レベルが減少している DNA」としては、以下の (a)〜（e)の!、ずれかに記載の DNAが挙げられる。  [0102] (8) In the trunk trunk of the two different plants (preferably the trunk of the trunk trunk or the middle of the trunk), the eucalyptus is compared with the xylem side of the trunk of the Eucalyptus globulus. The expression level of genus Camaldrensis is reduced at the xylem side of the trunk, and the expression level of at least one of the DNA is detected, and the expression level of the DNA in the trunk xylem of two different plants is detected. As a result of comparison, the expression level of the DNA is reduced in plant A compared to plant B. In the present invention, "the Eucalyptus genus Camaldrensis is compared with the xylem side of the trunk of Eucalyptus globules." Examples of the “DNA whose expression level is reduced on the xylem side of the trunk” include the following (a) to (e)!
(a)配列番号： 116に記載のアミノ酸配列力もなるタンパク質をコードする DNA (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 116
(b)配列番号： 105に記載の塩基配列を含む DNA (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 105
(c)配列番号： 116に記載のアミノ酸配列力もなるタンパク質と 50%以上の相同性を 有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in SEQ ID NO: 116
(d)配列番号： 105に記載の塩基配列からなる DNAとストリンジ ントな条件下でノ、ィ ブリダィズする DNA (d) DNA having the nucleotide sequence set forth in SEQ ID NO: 105 and DNA under stringent conditions DNA to be blinded
(e)配列番号： 116に記載のアミノ酸配列において 1または複数のアミノ酸が置換、欠 失、付加、および Zまたは挿入されたアミノ酸配列からなるタンパク質をコードする DN A  (e) DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence set forth in SEQ ID NO: 116
[0103] 本発明の検査方法を利用することで、上記（1)〜（8)のいずれかに該当する木部ま たは該木部を有する植物を選択し、選択された木部または該木部を有する植物から 均一なノルプを作製することが可能であり、パルプ製造の効率化、コストの削減、新 規な特性を持った紙の製造が可能となる。なお、以下の（1)〜（8)のいずれかに該 当する木部または該木部を有する植物を選択後、当業者に周知の方法に従って、選 択された木部間または該木部を有する植物間で木繊維状態が均一であるかを確認 した後に、パルプを作製することもできる。また、以下の（1)〜（8)のいずれかに該当 しな 、木部または該木部を有する植物を集めて、任意の配合のパルプを作製するこ とちでさる。  [0103] By using the inspection method of the present invention, a xylem corresponding to any of the above (1) to (8) or a plant having the xylem is selected, and the selected xylem or the xylem is selected. It is possible to produce a uniform norp from plants with xylem, making it possible to increase the efficiency of pulp production, reduce costs, and manufacture paper with new characteristics. In addition, after selecting a xylem corresponding to any of the following (1) to (8) or a plant having the xylem, between selected xylems or the xylem according to a method well known to those skilled in the art Pulp can also be produced after confirming whether the wood fiber state is uniform among plants having the. Further, if it does not fall under any of the following (1) to (8), it is obtained by collecting a xylem or a plant having the xylem to produce a pulp having an arbitrary composition.
[0104] 本発明の検査は、公知の種々の方法で実施することができる。例えば、まず、被検 植物の所望の組織から RNA試料を調製する。次いで、該 RNA試料に含まれるターゲ ット RNAの量を測定する。次いで、測定された RNAの量を対照と比較する。このような 方法としては、ノーザンブロッテイング法、または、 RT-PCR法等を例示することができ る。  [0104] The test of the present invention can be carried out by various known methods. For example, first, an RNA sample is prepared from a desired tissue of a test plant. Next, the amount of target RNA contained in the RNA sample is measured. The amount of RNA measured is then compared to a control. Examples of such methods include Northern blotting and RT-PCR.
[0105] 本発明の検査は、 DNAアレイ法によっても実施することができる。本発明において、 ヌクレオチドの固定 (アレイ）方法として、 Aflfymetrix社開発によるオリゴヌクレオチドを 基本としたアレイが例示できる。オリゴヌクレオチドのアレイにおいて、オリゴヌクレオ チドは通常 in situで合成される。例えば、 photolithographicの技術（Aflfymetrix社）、お よび化学物質を固定させるためのインクジェット (Rosetta Inpharmatics社)技術等によ るオリゴヌクレオチドのインサイチュ合成法が既に知られており、いずれの技術も本発 明の基板の作製に利用することができる。  [0105] The test of the present invention can also be performed by a DNA array method. In the present invention, examples of the nucleotide immobilization (array) method include an oligonucleotide-based array developed by Aflfymetrix. In an oligonucleotide array, oligonucleotides are usually synthesized in situ. For example, in-situ synthesis methods of oligonucleotides using photolithographic technology (Aflfymetrix) and inkjet (Rosetta Inpharmatics) technology for immobilizing chemical substances are already known. It can be used for the production of a substrate.
[0106] 本発明にお 、て「基板」とは、ヌクレオチドプローブを固定することが可能な板状の 材料を意味する。本発明の基板は、ヌクレオチドプローブを固定することができれば 特に制限はないが、一般に DNAアレイ技術で使用される基板を好適に用いることが できる。一般に DNAアレイは、高密度に基板にプリントされた何千ものヌクレオチドで 構成されている。通常これらの DNAは非透過性 (non- porous)の基板の表層にプリン トされる。基板の表層は、一般的にはガラスである力 透過性 (porous)の膜、例えば- トロセルロースメンブレムを使用することができる。 In the present invention, the “substrate” means a plate-like material on which a nucleotide probe can be fixed. The substrate of the present invention is not particularly limited as long as the nucleotide probe can be immobilized, but a substrate generally used in DNA array technology is preferably used. it can. In general, a DNA array consists of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate. As the surface layer of the substrate, a force permeable membrane, which is generally glass, can be used, for example, a trocellulose membrane.
[0107] 基板に固定するヌクレオチドプローブは、本発明の DNAの発現を検出することがで きるものであれば、特に制限されない。即ち該プローブは、本発明の DNAにハイブリ ダイズするようなプローブである。特異的なハイブリダィズが可能であれば、ヌクレオ チドプローブは、本発明の DNAに対し、完全に相補的である必要はない。本発明に おいて基板に結合させるヌクレオチドプローブの長さは、特に制限されないが、通常 10〜100bpであり、好ましくは 10〜50bpであり、さらに好ましくは 15〜25bpである。  [0107] The nucleotide probe immobilized on the substrate is not particularly limited as long as it can detect the expression of the DNA of the present invention. That is, the probe is a probe that hybridizes to the DNA of the present invention. If specific hybridization is possible, the nucleotide probe need not be perfectly complementary to the DNA of the present invention. In the present invention, the length of the nucleotide probe to be bound to the substrate is not particularly limited, but is usually 10 to 100 bp, preferably 10 to 50 bp, more preferably 15 to 25 bp.
[0108] 本方法においては、本発明の DNAと該基板を接触させる。この過程により、上記ヌ クレオチドプローブに対し、本発明の DNAをハイブリダィズさせる。ハイブリダィゼー シヨンの反応液および反応条件は、基板に固定するヌクレオチドプローブの長さ等の 諸要因により変動しうるが、一般的に当業者に周知の方法により行うことができる。  [0108] In the present method, the DNA of the present invention is brought into contact with the substrate. Through this process, the DNA of the present invention is hybridized to the nucleotide probe. Although the hybridization reaction solution and reaction conditions may vary depending on various factors such as the length of the nucleotide probe immobilized on the substrate, it can be generally performed by methods well known to those skilled in the art.
[0109] 本方法においては、次いで、本発明の DNAと該基板に固定されたヌクレオチドプロ ーブとのハイブリダィズの強度を検出する。この検出は、例えば、本発明の DNAに標 識した蛍光色素の蛍光シグナルをスキャナ一等によって読み取ることによって行うこ とがでさる。  [0109] In the present method, the intensity of hybridization between the DNA of the present invention and the nucleotide probe immobilized on the substrate is then detected. This detection can be performed, for example, by reading the fluorescence signal of the fluorescent dye labeled on the DNA of the present invention with a scanner or the like.
[0110] また、本発明は、本発明の DNAのプロモーター DNAを提供する。本発明のプロモ 一ター DNAとしては、本発明によって得られた植物、特に榭木の空間的位置におい て発現差のある遺伝子に連なるプロモーター DNAが挙げられる。ここで、「プロモータ 一 DNA」とは、 DNAを铸型とした mRNAの合成 (転写）の開始に必要な特定塩基配列 を含む DNAを意味し、これには自然界に存在する DNAの他、組換えなどの人工的な 改変操作により作成された DNAが含まれる。  [0110] The present invention also provides a promoter DNA of the DNA of the present invention. The promoter DNA of the present invention includes a promoter DNA linked to a gene obtained by the present invention, in particular, a gene having a differential expression in the spatial position of an oak. Here, “promoter-one DNA” means DNA containing a specific base sequence necessary for the initiation of synthesis (transcription) of mRNA using DNA as a saddle type. It includes DNA created by artificial alterations such as replacement.
[0111] 本発明のプロモータ DNAとしては、より具体的には、植物樹幹の木繊維細胞壁を形 成する機能を有するタンパク質 (セルロース合成に関与するタンパク質、およびリグ- ン合成に関与するタンパク質）をコードする DNAのプロモーター DNA、及び植物の転 写因子をコードする DNAのプロモーター DNAが挙げられる。植物樹幹の木繊維細胞 壁を形成する機能を有するタンパク質 (セルロース合成に関与するタンパク質、およ びリグニン合成に関与するタンパク質）をコードする DNAのプロモーター DNAを配列 番号： 93〜： LOOに示す。植物樹幹の木繊維細胞壁を形成する機能を有するタンパク 質 (セルロース合成に関与するタンパク質、およびリグニン合成に関与するタンパク質[0111] More specifically, the promoter DNA of the present invention includes a protein having a function of forming a tree fiber cell wall of a plant trunk (a protein involved in cellulose synthesis and a protein involved in ligne synthesis). Examples include promoter DNA of encoding DNA and promoter DNA of DNA encoding plant transcription factor. Plant trunk tree fiber cells The promoter DNA of DNA encoding a protein having a function of forming a wall (a protein involved in cellulose synthesis and a protein involved in lignin synthesis) is shown in SEQ ID NO: 93-: LOO. Proteins that have the function of forming tree fiber cell walls of plant trunks (proteins involved in cellulose synthesis and proteins involved in lignin synthesis)
)をコードする DNAと、そのプロモーター DNAとの関係は、実施例に記載の通りである 。また、植物の転写因子をコードする DNAのプロモーター DNAを、配列番号： 156〜 166に示す。植物の転写因子をコードする DNAとそのプロモータ DNAの関係もまた、 実施例に記載の通りである。 The relationship between the DNA encoding) and its promoter DNA is as described in the Examples. Further, promoter DNAs of DNAs encoding plant transcription factors are shown in SEQ ID NOs: 156 to 166. The relationship between DNA encoding a plant transcription factor and its promoter DNA is also as described in the Examples.
[0112] 本発明のプロモーターは、例えば、以下のようにして製造し、利用することができる 。 目的とするユーカリ植物の組織より DNAを抽出し精製する。 DNAの調製に際しては 、各種の方法を用いることができ、市販のキット、例えば、 ISOPLANTキット（二ツボン ジーン社製)などを用いることが可能である。  [0112] The promoter of the present invention can be produced and used as follows, for example. Extract and purify DNA from the tissues of the desired eucalyptus plant. In preparing DNA, various methods can be used, and commercially available kits such as ISOPLANT kit (manufactured by Futtsubon Gene Co., Ltd.) can be used.
[0113] 得られた DNAを材料として、本発明者らが既に単離に成功しているユーカリ cDNA の塩基配列を基に、任意の 2箇所よりオリゴヌクレオチドを作製し、これをプライマーに 用いた PCR法により、選んだユーカリ cDNAに対応するゲノム DNAを簡単に製造する ことが可能である。該遺伝子の上流域 DNAは、該遺伝子の塩基配列を基に作成した オリゴヌクレオチドプライマーを利用する PCR法（Inverse- PCR法やアンカー PCR法 ·Τ AIL-PCR法（島本功ら監修、「新版植物の PCR実験プロトコール」（植物細胞工学別 冊、植物細胞工学シリーズ 7)秀潤社 1997年 7月発行）、あるいは該遺伝子の DNA配 列をプローブに用いるハイブリダィゼ—シヨン法により、単離することが可能である。  [0113] Using the obtained DNA as a material, oligonucleotides were prepared from any two locations based on the base sequence of eucalyptus cDNA that had been successfully isolated by the present inventors and used as primers. By PCR, genomic DNA corresponding to the selected eucalyptus cDNA can be easily produced. Upstream DNA of the gene is obtained by PCR using oligonucleotide primers created based on the base sequence of the gene (Inverse-PCR method or anchor PCR method · AIL-PCR method (supervised by Isao Shimamoto, PCR Experiment Protocol ”(plant cell engineering separate volume, plant cell engineering series 7) published by Shujunsha, July 1997), or by the hybridization method using the DNA sequence of the gene as a probe. Is possible.
[0114] ユーカリの DNAには、ゲノム DNAライブラリーを利用することも可能である。ゲノム D ΝΑライブラリ一は、ユーカリから抽出した DNAをえ DNA由来の各種ベクターの他、コ スミドベクター、 TACベクター（Liuら（1999)、 Proc. Natl. Acad. Sci. USA, vol96： p653 5)などのクローユングベクターに挿入し、大腸菌を形質転換して得られる。  [0114] A genomic DNA library can also be used for eucalyptus DNA. In addition to DNA extracted from eucalyptus, various genome vectors, TAC vectors (Liu et al. (1999), Proc. Natl. Acad. Sci. USA, vol96: p653 5) It is obtained by transforming E. coli.
[0115] ゲノム DNAライブラリーのスクリーニングには、ハイブリダィゼーシヨン技術を用いる ことができる。プローブには、本発明者らが既に単離に成功しているユーカリ cDNAの 配列を用いることができる。このプローブを用いて上記のゲノム DNAライブラリーをス クリーニングして、該遺伝子と相同な DNA配列が含まれるクローンを単離する。制限 酵素切断地図の作成や、塩基配列の決定などを行い、クローンィ匕された DNAの構造 を明らかにし、該遺伝子の上流に存在する配列を特定する。この上流配列は TATA ボックス配列を含み、少なくとも数百 bp力も数 kbpの大きさのものであることが望ましい 。この配列を適当な制限酵素によって切り出し、必要に応じて他のプラスミドベクター 等にサブクロー-ングする。 [0115] Hybridization technology can be used for screening genomic DNA libraries. As the probe, the eucalyptus cDNA sequence that has been successfully isolated by the present inventors can be used. Using this probe, the above genomic DNA library is screened to isolate a clone containing a DNA sequence homologous to the gene. Restriction Enzyme cleavage maps are created and nucleotide sequences are determined, the structure of the cloned DNA is clarified, and the sequence existing upstream of the gene is identified. This upstream sequence preferably includes a TATA box sequence and is at least several hundred bp force of several kbp. This sequence is excised with an appropriate restriction enzyme and subcloned into another plasmid vector or the like as necessary.
[0116] 上記の配列のプロモーター活性については、以下のようにして解析することが可能 である。例えば、 ρΒΙΙΟΙなどレポーター遺伝子を含むベクターを用い、そのレポータ 一遺伝子の上流に上記の配列を連結するようにサブクローユングする。 ρΒΙΙΟΙベクタ 一にはレポーター遺伝子として、大腸菌の /3ダルク口-ダーゼ (GUS)が使用されて いる。この遺伝子産物は基質として 5- bromo- 4- chloro- 3- j8 - D- glucronic acid (X- glu c)を用いると、これを分解して青色の沈澱物である indigotinを生ずるため、遺伝子発 現を組織レベルでモニターすることが可能である。また、基質として 4- methyHimbellif ery卜 j8 - D- glucronide (4MUG)を用いると、遺伝子産物の働きによって生じる蛍光に よって遺伝子発現を定量することが可能である。なお、レポーター遺伝子としては、 G US遺伝子の他にクロラムフエ-コールァセチルトランスフェラーゼ遺伝子や、ルシフ エラーゼ遺伝子、グリーンフルォロセインプロテイン遺伝子なども利用が可能である。  [0116] The promoter activity of the above sequence can be analyzed as follows. For example, using a vector containing a reporter gene such as ρΒΙΙΟΙ, subcloning is performed so that the above sequence is linked upstream of the reporter gene. The ρΒΙΙΟΙ vector uses E. coli / 3 dalc mouth-dase (GUS) as a reporter gene. When this gene product uses 5-bromo-4-chloro-3-j8-D-glucronic acid (X-glucuric acid) as a substrate, it decomposes to produce indigotin, a blue precipitate. It is possible to monitor the current situation at the organizational level. When 4-methyHimbellif eryj8-D-glucronide (4MUG) is used as a substrate, gene expression can be quantified by fluorescence generated by the action of the gene product. In addition to the GUS gene, a chloramphee-cholacetyl transferase gene, a luciferase gene, a green fluorescein protein gene, and the like can be used as a reporter gene.
[0117] 上記のようにして作成されたキメラ遺伝子構築物は、例えば、ァグロバタテリゥムを 介してシロイヌナズナなどの植物に導入してその機能を解析することが可能である。 p BI101をベクターとして用いた場合は、キメラ遺伝子を含む組換えプラスミドを、例え ば、ァグロバタテリゥム 'ッメファシエンスの MP90株にエレクト口ポレーシヨン法を用い て導入し、得られた形質転換菌を、例えば、フローラルディップ法（島本功ら監修、「 モデル植物の実験プロトコール」（植物細胞工学別冊、植物細胞工学シリーズ 4)秀 潤社 1996年 4月発行）によりシロイヌナズナ植物体に感染させる。感染処理した植物 より得られた種子を、用いたベクターに基づ ヽてカナマイシンなどの薬剤を含む培地 に播種し、遺伝子導入により薬剤耐性となった形質転 物体を得る。この形質転 ^¾物体を用いてレポーター GUS遺伝子の発現について解析する。本発明のプロ モーターまたはそれを含む発現ベクターは、以下のようにして利用することが可能で ある。本発明のプロモーターの下流に目的の遺伝子、例えば、ある種の二次代謝産 物生合成に関わる遺伝子を連結したキメラ遺伝子を、例えば、 ρΒΙΙΟΙベクターに挿 入し発現ベクターを構築する。このベクターをァグロバタテリゥムを介して、例えば、タ バコ植物体に導入する。得られた形質転換植物においては、本発明のプロモーター の働きにより、該遺伝子が木繊維形成部位に発現し、任意の二次代謝物生産が可 能となるものと期待される。この場合、 35Sプロモーターのように不要な組織において も発現する現象がな 、ため、他の好ましくな 、形質が現れな 、ことが期待される。 [0117] The chimeric gene construct prepared as described above can be introduced into plants such as Arabidopsis thaliana via agrobacterium and analyzed for its function. When pBI101 is used as a vector, a recombinant plasmid containing the chimeric gene is introduced into the MP90 strain of Agrobacterium tumefaciens, for example, using the electoporation method, and the resulting transformant For example, Arabidopsis thaliana plants are infected by the floral dip method (supervised by Isao Shimamoto et al., "Experimental protocol for model plants" (plant cell engineering separate volume, plant cell engineering series 4) Shujunsha published in April 1996). Seeds obtained from the infected plant are sown in a medium containing a drug such as kanamycin based on the vector used to obtain a transformant that has become drug-resistant by gene transfer. Using this transformed substance, the expression of the reporter GUS gene is analyzed. The promoter of the present invention or an expression vector containing the promoter can be used as follows. The gene of interest downstream of the promoter of the present invention, such as certain secondary metabolites An expression vector is constructed by inserting a chimeric gene in which genes involved in physical biosynthesis are linked, for example, into a ρΒΙΙΟΙ vector. This vector is introduced into, for example, Tabacco plants via agrobacterium. In the resulting transformed plant, the gene of the present invention is expected to be expressed at the site of tree fiber formation and to produce any secondary metabolite by the action of the promoter of the present invention. In this case, since there is no phenomenon that is expressed even in an unnecessary tissue like the 35S promoter, it is expected that other preferable traits do not appear.
[0118] 本発明のプロモーターで制御可能な遺伝子としては、上述した特定の遺伝子に限 定されない。また、本発明のプロモーターに他の発現制御配列を連結して本発明の プロモーターの機能を改変することも可能である。このような発現制御配列としては、 ェンハンサー配列ゃレプレッサー配列、インスレーター配列などが挙げられる。本発 明のプロモーターには、機能特性として、榭幹の地上高位置特異的ならびに細胞壁 生合成にカゝかる遺伝子の発現を制御する幾つかのシスエレメント配列が含まれる。本 発明のプロモーターに含まれるシスエレメント配列の利用を目的として、他のプロモ 一ターに本発明のプロモーターの一部を挿入連結し、そのプロモーターの機能を改 変することも可能である。  [0118] The gene that can be controlled by the promoter of the present invention is not limited to the specific gene described above. It is also possible to modify the function of the promoter of the present invention by linking other expression control sequences to the promoter of the present invention. Examples of such expression control sequences include enhancer sequences, repressor sequences, and insulator sequences. The promoter of the present invention includes several cis-element sequences that control the expression of genes involved in the above-ground specific location of trunk and cell wall biosynthesis as functional characteristics. For the purpose of using the cis element sequence contained in the promoter of the present invention, it is possible to insert a part of the promoter of the present invention into another promoter and modify the function of the promoter.
[0119] また、本発明は、植物の木繊維細胞壁形成に関わるタンパク質をコードする DNAの 発現を抑制するための DNAを提供する。内在性遺伝子の発現を抑制するための DN Aの好まし 、態様としては、本発明の DNAの転写産物と相補的なアンチセンス RNAを コードする DNA、本発明の DNAの転写産物を特異的に開裂するリボザィム活性を有 する RNAをコードする DNA、 RNAi効果または共抑制効果により、本発明の DNAの発 現を抑制する RNAをコードする DNA、本発明の DNAの転写産物に対してドミナントネ ガティブな形質を有するタンパク質をコードする DNA等を例示することができる。上記 「内在性遺伝子の発現抑制」には、遺伝子の転写の抑制、および Zまたは該遺伝子 力もコードされるタンパク質への翻訳の抑制が含まれる。また、該遺伝子の発現の完 全な停止のみならず発現の減少も含まれる。  [0119] The present invention also provides DNA for suppressing the expression of a DNA encoding a protein involved in plant fiber fiber wall formation. The use of DNA for suppressing the expression of an endogenous gene is preferred in that the DNA encoding an antisense RNA complementary to the transcription product of the DNA of the present invention and the transcription product of the DNA of the present invention are specifically used. RNA encoding RNA with ribozyme activity to cleave, DNA encoding RNA that suppresses expression of DNA of the present invention by RNAi effect or co-suppression effect, dominant negative for transcript of DNA of the present invention Examples thereof include DNA encoding a protein having various traits. The above “suppression of endogenous gene expression” includes suppression of gene transcription and suppression of translation into Z or a protein that also encodes the gene force. Also included is a decrease in expression as well as complete cessation of expression of the gene.
[0120] 植物における特定の内在性遺伝子の発現を抑制する方法としては、アンチセンス 技術を利用する方法が当業者に最もよく利用されている。植物細胞におけるアンチ センス効果は、電気穿孔法で導入したアンチセンス RNAが植物にお!、てアンチセン ス効果を発揮することをエッカーらが示したことで初めて実証された (Ecker, JR. & Da vis, RW" Proc Natl Acad Sci USA, 1986, 83, 5372.)。その後、タバコやペチュニア においてもアンチセンス RNAの発現により標的遺伝子の発現が低下した例が報告さ れており（van der Krol AR. et al.， Nature, 1988, 333, 866.)、現在では、アンチセン ス技術は植物における遺伝子発現を抑制させる手段として確立している。 [0120] As a method for suppressing the expression of a specific endogenous gene in a plant, a method using an antisense technique is most often used by those skilled in the art. The antisense effect in plant cells is that antisense RNA introduced by electroporation is added to plants! (Ecker, JR. & Da vis, RW "Proc Natl Acad Sci USA, 1986, 83, 5372.) Later, in tobacco and petunia An example of a decrease in target gene expression due to the expression of antisense RNA has been reported (van der Krol AR. Et al., Nature, 1988, 333, 866.). At present, antisense technology is applied to genes in plants. Established as a means to suppress expression.
[0121] アンチセンス核酸が標的遺伝子の発現を抑制する作用としては、以下のような複数 の要因が存在する。すなわち、三重鎖形成による転写開始阻害、 RNAポリメラーゼに よって局部的に開状ループ構造が作られた部位とのハイブリッド形成による転写阻害 、合成の進みつつある RNAとのハイブリッド形成による転写阻害、イントロンとェキソン との接合点におけるハイブリッド形成によるスプライシング阻害、スプライソソーム形成 部位とのハイブリッド形成によるスプライシング阻害、 mRNAとのハイブリッド形成によ る核から細胞質への移行阻害、キヤッビング部位やポリ (A)付加部位とのハイブリッド 形成によるスプライシング阻害、翻訳開始因子結合部位とのハイブリッド形成による 翻訳開始阻害、開始コドン近傍のリボソーム結合部位とのハイブリッド形成による翻 訳阻害、 mRNAの翻訳領域やポリソーム結合部位とのハイブリッド形成によるペプチド 鎖の伸長阻害、および核酸とタンパク質との相互作用部位とのハイブリッド形成によ る遺伝子発現阻害などである。このようにアンチセンス核酸は、転写、スプライシング または翻訳など様々な過程を阻害することで、標的遺伝子の発現を抑制する (平島 および井上，新生化学実験講座 2核酸 IV遺伝子の複製と発現， 日本生化学会編， 東京化学同人， 1993, 319-347.) o  [0121] There are several factors as described below for the action of an antisense nucleic acid to suppress the expression of a target gene. Inhibition of transcription initiation by triplex formation, inhibition of transcription by hybridization with a site where an open loop structure was locally created by RNA polymerase, inhibition of transcription by hybridization with RNA that is undergoing synthesis, intron and Splicing inhibition by hybridization at the junction with exon, splicing inhibition by hybridization with spliceosome formation site, inhibition of translocation from nucleus to cytoplasm by hybridization with mRNA, capping site and poly (A) addition site Splicing inhibition by hybridization, translation initiation inhibition by hybridization with a translation initiation factor binding site, translation inhibition by hybridization with a ribosome binding site near the initiation codon, hybridization with mRNA translation region and polysome binding site Elongation inhibition of that peptide chain, and the like by that gene expression inhibition hybridization to the interaction site between a nucleic acid and a protein. In this way, antisense nucleic acids suppress the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kogaku 2 Nucleic acid IV gene replication and expression, Japan biochemicalization) The Society, Tokyo Chemistry, 1993, 319-347.) O
[0122] 本発明で用いられるアンチセンス配列は、上記のいずれの作用により標的遺伝子 の発現を抑制してもよい。一つの態様としては、遺伝子の mRNAの 5'端近傍の非翻訳 領域に相補的なアンチセンス配列を設計すれば、遺伝子の翻訳阻害に効果的と考 えられる。また、コード領域もしくは 3'側の非翻訳領域に相補的な配列も使用すること ができる。このように、遺伝子の翻訳領域だけでなく非翻訳領域の配列のアンチセン ス配列を含む DNAも、本発明で利用されるアンチセンス DNAに含まれる。使用される アンチセンス DNAは、適当なプロモーターの下流に連結され、好ましくは 3'側に転写 終結シグナルを含む配列が連結される。このようにして調製された DNAは、公知の方 法を用いることで、所望の植物へ形質転換できる。アンチセンス DNAの配列は、形質 転換される植物が持つ内在性遺伝子またはその一部と相補的な配列であることが好 ましいが、遺伝子の発現を有効に抑制できる限りにおいて、完全に相補的でなくても よい。転写された RNAは、標的遺伝子の転写産物に対して好ましくは 90%以上、最も 好ましくは 95%以上の相補性を有する。アンチセンス配列を用いて標的遺伝子の発 現を効果的に抑制するには、アンチセンス DNAの長さは少なくとも 15塩基以上であり 、好ましくは 100塩基以上であり、さらに好ましくは 500塩基以上である。通常用いられ るアンチセンス DNAの長さは 5kbよりも短ぐ好ましくは 2.5kbよりも短い。 [0122] The antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above. In one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of a gene is designed, it is considered effective for inhibiting the translation of the gene. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, DNA containing an antisense sequence of not only a translation region of a gene but also an untranslated region is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The DNA prepared in this way can be By using the method, it can be transformed into a desired plant. The sequence of the antisense DNA is preferably a sequence complementary to an endogenous gene or a part of the plant to be transformed, but is completely complementary as long as the gene expression can be effectively suppressed. It doesn't have to be. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene. In order to effectively suppress the expression of a target gene using an antisense sequence, the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. . The length of the antisense DNA usually used is shorter than 5 kb, preferably shorter than 2.5 kb.
[0123] また、内在性遺伝子の発現の抑制は、リボザィム、またはリボザィムをコードする DN Aを利用して行うことも可能である。リボザィムとは触媒活性を有する RNA分子のことを 指す。リボザィムには種々の活性を有するものが存在する力 中でも RNAを切断する 酵素としてのリボザィムに焦点を当てた研究により、 RNAを部位特異的に切断するリ ボザィムの設計が可能となった。リボザィムには、グループ Iイントロン型や RNase Pに 含まれる Ml RNAのように 400ヌクレオチド以上の大きさのものもあるが、ハンマーへッ ド型ゃヘアピン型と呼ばれる 40ヌクレオチド程度の活性ドメインを有するものもある（ 小泉誠および大塚栄子，蛋白質核酸酵素， 1990, 35, 2191.)。  [0123] In addition, suppression of endogenous gene expression can also be performed using ribozymes or DNA encoding ribozymes. Ribozyme refers to an RNA molecule that has catalytic activity. Among the abilities of ribozymes having various activities, research focused on ribozymes as enzymes that cleave RNA, among others, has enabled the design of ribozymes that cleave RNA in a site-specific manner. Some ribozymes have a size of 400 nucleotides or more, such as the group I intron type and Ml RNA contained in RNase P, but the hammerhead type has an active domain of about 40 nucleotides called the hairpin type. There are also (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191.).
[0124] 例えば、ハンマーヘッド型リボザィムの自己切断ドメインは、 G13U14C15という配列 の C15の 3'側を切断する力 その活性には U14と A9との塩基対形成が重要とされ、 C1 5の代わりに A15または U15でも切断され得ることが示されている（Koizumi, M. et al., FEBS Lett, 1988, 228, 228.) 0基質結合部位が標的部位近傍の RNA配列と相補的 なリボザィムを設計すれば、標的 RNA中の UC、 UUまたは UAという配列を認識する制 限酵素的な RNA切断リボザィムを作出することができる（Koizumi, M. et al., FEBS Le tt, 1988, 239, 285.、小泉誠および大塚栄子，蛋白質核酸酵素， 1990, 35, 2191.、 Ko izumi, M. et al., Nucl Acids Res, 1989, 17, 7059.) 0例えば、植物体の木繊維細胞壁 形成に関わるタンパク質をコードする DNAのコード領域中には、標的となり得る部位 が複数存在する。 [0124] For example, the self-cleaving domain of the hammerhead ribozyme has the ability to cleave the 3 'side of C15 in the sequence G13U14C15. For its activity, base pairing between U14 and A9 is important. It has been shown that A15 or U15 can also be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.) 0 Design a ribozyme whose substrate binding site is complementary to the RNA sequence near the target site Thus, a restriction enzyme-like RNA cleavage ribozyme that recognizes the sequence UC, UU, or UA in the target RNA can be generated (Koizumi, M. et al., FEBS Lett, 1988, 239, 285. , Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191., Koizumi, M. et al., Nucl Acids Res, 1989, 17, 7059.) 0 For example, involved in the formation of wood fiber cell walls in plants There are multiple sites that can be targeted in the coding region of the DNA encoding the protein.
[0125] また、ヘアピン型リボザィムも本発明の目的に有用である。このリボザィムは、例え ばタバコリングスポットウィルスのサテライト RNAのマイナス鎖に見出される（Buzayan, JM., Nature, 1986, 323, 349.)。ヘアピン型リボザィムからも、標的特異的な RNA切断 リボザィムを作出できることが示されている（Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751.、菊池洋，化学と生物， 1992, 30, 112.)。 [0125] Hairpin ribozymes are also useful for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that target-specific RNA cleavage ribozymes can also be generated from hairpin ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.).
[0126] 標的を切断できるように設計されたリボザィムは、植物細胞中で転写されるように、 カリフラワーモザイクウィルスの 35Sプロモーターなどのプロモーターおよび転写終結 配列に連結される。このとき、転写された RNAの 5'端や 3'端に余分な配列が付加され ていると、リボザィムの活性が失われることがある力 こういった場合は、転写されたリ ボザィムを含む RNAからリボザィム部分だけを正確に切り出すために、リボザィム部 分の 5'側や 3'側にシスに働く別のトリミングリボザィムを配置させることも可能である (T aira, K. et al, Protein Eng, 1990, 3, 733.、 Dzianott, AM. & Bujarski, JJ.， Proc Natl Acad Sci USA, 1989, 86, 4823.、 Grosshans, CA. & Cech, TR., Nucl Acids Res, 199 1, 19, 3875.、 Taira, K. et al" Nucl Acids Res, 1991, 19, 5125.)。また、このような構 成単位をタンデムに並べ、標的遺伝子内の複数の部位を切断できるようにすることで 、より効果を高めることもできる (Yuyama, N. et al" Biochem Biophys Res Commun, 1 992, 186, 1271.)。このように、リボザィムを用いて本発明における標的遺伝子の転写 産物を特異的に切断することで、該遺伝子の発現を抑制することができる。  [0126] The ribozyme designed to cleave the target is linked to a promoter and transcription termination sequence, such as the cauliflower mosaic virus 35S promoter, so that it is transcribed in plant cells. At this time, if an extra sequence is added to the 5 'or 3' end of the transcribed RNA, the activity of the ribozyme may be lost. In this case, the RNA containing the transcribed ribozyme It is possible to place another trimming ribozyme that acts on cis on the 5 'side or 3' side of the ribozyme part (T aira, K. et al, Protein Eng, 1990, 3, 733., Dzianott, AM. & Bujarski, JJ., Proc Natl Acad Sci USA, 1989, 86, 4823., Grosshans, CA. & Cech, TR., Nucl Acids Res, 199 1, 19, 3875., Taira, K. et al "Nucl Acids Res, 1991, 19, 5125.) Also, such structural units are arranged in tandem so that multiple sites in the target gene can be cleaved. Thus, the effect can be further enhanced (Yuyama, N. et al "Biochem Biophys Res Commun, 1 992, 186, 1271.). Thus, by specifically cleaving the transcription product of the target gene in the present invention using a ribozyme, the expression of the gene can be suppressed.
[0127] 内在性遺伝子の発現の抑制は、さらに、標的遺伝子配列と同一もしくは類似した配 列を有する二本鎖 RNAを用いた RNA干渉（RNA interferance； RNAi)によっても行うこ とができる。 RNAiとは、標的遺伝子配列と同一もしくは類似した配列を有する二重鎖 RNAを細胞内に導入すると、導入した外来遺伝子および標的内在性遺伝子の発現 カ^、ずれも抑制される現象のことを指す。 RNAiの機構の詳細は明らかではな 、が、 最初に導入した二本鎖 RNAが小片に分解され、何らかの形で標的遺伝子の指標と なること〖こより、標的遺伝子が分解されると考えられている。 RNAiは植物においても 効果を奏することが知られている（Chuang, CF. & Meyerowitz, EM., Proc Natl Acad Sci USA, 2000, 97, 4985.) 0例えば、ユーカリ種間の木繊維性質の違いを制御するタ ンパク質をコードする DNAの発現を RNAiにより抑制するためには、配列番号： 1〜34 のいずれかに記載の塩基配列、または、これらと類似した配列を有する二本鎖 RNA を目的の植物へ導入すればよい。 RNAiに用いる遺伝子は、標的遺伝子と完全に同 一である必要はないが、少なくとも 70%以上、好ましくは 80%以上、さらに好ましくは 9 0%以上、最も好ましくは 95%以上の配列の同一性を有する。また、配列の同一性は 上述した手法により決定できる。 [0127] Inhibition of endogenous gene expression can also be performed by RNA interference (RNAi) using double-stranded RNA having the same or similar sequence as the target gene sequence. RNAi refers to a phenomenon in which the expression of the introduced foreign gene and target endogenous gene and the deviation are suppressed when a double-stranded RNA having the same or similar sequence as the target gene sequence is introduced into the cell. . The details of the RNAi mechanism are not clear, but it is thought that the target gene is degraded from the fact that the double-stranded RNA introduced first is broken down into small pieces and somehow serves as an indicator of the target gene. . RNAi is known to be effective in plants (Chuang, CF. & Meyerowitz, EM., Proc Natl Acad Sci USA, 2000, 97, 4985.) 0 For example, differences in wood fiber properties among eucalyptus species In order to suppress the expression of a DNA encoding a protein that regulates protein by RNAi, a double-stranded RNA having a nucleotide sequence set forth in any of SEQ ID NOs: 1-34 or a sequence similar thereto is used. What is necessary is just to introduce | transduce into the target plant. The gene used for RNAi is completely the same as the target gene. The sequence identity is at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more. Further, the identity of the sequence can be determined by the method described above.
[0128] 内在性遺伝子の発現の抑制は、標的遺伝子配列と同一もしくは類似した配列を有 する DNAの形質転換によって起こる共抑制によっても達成できる。「共抑制」とは、植 物に標的内在性遺伝子と同一もしくは類似した配列を有する遺伝子を形質転換によ り導入すると、導入した外来遺伝子および標的内在性遺伝子の発現がいずれも抑制 される現象のことを指す。共抑制の機構の詳細は明らかではないが、少なくともその 機構の一部は RNAiの機構と重複して 、ると考えられて 、る。共抑制も植物にお!、て 観察される（Smyth, DR., Curr Biol, 1997, 7, R793.、 Martienssen, R.， Curr Biol, 199 6, 6, 810.)。例えば、植物体における木繊維形成を制御するタンパク質をコードする DNAが共抑制された植物体を得るためには、該 DNA、または、これらと類似した配列 を有する DNAを発現できるように作製したベクター DNAを目的の植物へ形質転換す ればよい。共抑制に用いる遺伝子は、標的遺伝子と完全に同一である必要はないが 、少なくとも 70%以上、好ましくは 80%以上、さらに好ましくは 90%以上、最も好ましく は 95%以上の配列の同一性を有する。また、配列の同一性は上述した手法により決 定できる。 [0128] Suppression of endogenous gene expression can also be achieved by co-suppression caused by transformation of DNA having the same or similar sequence as the target gene sequence. “Co-suppression” is a phenomenon in which when a gene having the same or similar sequence as a target endogenous gene is introduced into a plant by transformation, the expression of the introduced foreign gene and the target endogenous gene are both suppressed. Refers to that. The details of the mechanism of co-suppression are not clear, but at least part of the mechanism is thought to overlap with the RNAi mechanism. Co-suppression is also observed in plants! (Smyth, DR., Curr Biol, 1997, 7, R793., Martienssen, R., Curr Biol, 199 6, 6, 810.). For example, in order to obtain a plant body in which DNA encoding a protein that controls the formation of wood fibers in the plant body is co-suppressed, a vector prepared so that the DNA or a DNA having a similar sequence can be expressed. Transform DNA into the target plant. The gene used for co-suppression need not be completely identical to the target gene, but at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more of sequence identity. Have. The sequence identity can be determined by the method described above.
[0129] さらに、本発明における内在性遺伝子の発現の抑制は、標的遺伝子がコードする タンパク質に対してドミナントネガティブの形質を有するタンパク質をコードする遺伝 子を、植物へ形質転換することによつても達成することができる。「ドミナントネガティ ブの形質を有するタンパク質をコードする遺伝子」とは、該遺伝子を発現させることに よって、植物体が本来持つ内在性の野生型タンパク質の活性を消失もしくは低下さ せる機能を有する遺伝子のことを指す。  [0129] Furthermore, the suppression of the expression of the endogenous gene in the present invention can also be achieved by transforming a gene encoding a protein having a dominant negative trait to the protein encoded by the target gene into a plant. Can be achieved. “A gene encoding a protein having a dominant negative trait” refers to a gene having a function of eliminating or reducing the activity of an endogenous wild-type protein inherent in a plant by expressing the gene. Refers to that.
[0130] また、本発明は、上記 DNAを含む組換えベクターを提供する。本発明のベクターと しては、植物細胞で転写可能なプロモーター配列と転写産物の安定ィヒに必要なポリ アデ-レーシヨン部位を含むターミネータ一配列を含んでいれば特に制限されず、 例えば、 pUC誘導体などの大腸菌で増幅可能なベクター、 ρΒΙΙΟΙ (クロンテック社)な どのように大腸菌とァグロバタテリゥムの双方で増幅可能なシャトルベクターなどが挙 げられる。また、植物ウィルス、例えば、カリフラワーモザイクウィルスをベクターとして 禾 IJ用することちでさる。 [0130] The present invention also provides a recombinant vector containing the DNA. The vector of the present invention is not particularly limited as long as it contains a promoter sequence that can be transcribed in plant cells and a terminator sequence that includes a polyadenylation site necessary for the stability of the transcript. For example, pUC Vectors that can be amplified in E. coli, such as derivatives, and shuttle vectors that can be amplified in both E. coli and agrobacterium, such as ρΒΙΙΟΙ (Clontech). I can get lost. Also, plant viruses such as cauliflower mosaic virus can be used as vectors for 禾 IJ.
[0131] 本発明のベクターは、例えば、ベクターの所定の部分に本発明のプロモーター DN Aや所望の遺伝子を恒常的または誘導的に発現させるためのプロモーター DNAを結 合あるいは挿入して得ることができる。なお、プロモーターをベクターに挿入する方法 は、通常の遺伝子をベクターに挿入する方法に従う。この組換えベクターのプロモー ターに所望の遺伝子を機能的に接続することで遺伝子発現用の発現ベクターを得る ことができる。  [0131] The vector of the present invention can be obtained, for example, by binding or inserting the promoter DNA of the present invention or a promoter DNA for constitutively or inducibly expressing a desired gene into a predetermined part of the vector. it can. The method for inserting the promoter into the vector follows the method for inserting a normal gene into the vector. An expression vector for gene expression can be obtained by functionally connecting a desired gene to the promoter of this recombinant vector.
[0132] 恒常的に発現させるためのプロモーターとしては、例えば、カリフラワーモザイクウイ ルスの 35Sプロモーター（Odell et al.， Nature, 1985, 313, 810.)、イネのァクチンプロ モーター（Zhang et al.， Plant Cell, 1991, 3, 1155.)、トウモロコシのュビキチンプロモ 一ター（Cornejo et al.， Plant Mol. Biol, 1993, 23, 567.)などが挙げられる。また、誘 導的に発現させるためのプロモーターとしては、例えば糸状菌 ·細菌 ·ウィルスの感 染ゃ侵入、低温、高温、乾燥、紫外線の照射、特定の化合物の散布などの外因によ つて発現することが知られて 、るプロモーターなどが挙げられる。このようなプロモー ターとしては、例えば、糸状菌'細菌'ウィルスの感染や侵入によって発現するイネキ チナーゼ遺伝子のプロモーター（Xu et al.， Plant Mol. Biol. , 1996, 30, 387.)やタパ コの PRタンパク質遺伝子のプロモーター（Ohshima et al.， Plant Cell, 1990, 2, 95.)、 低温によって誘導されるイネの「lipl9」遺伝子のプロモーター（Aguan et al.， Mol. Ge n Genet., 1993, 240,1.)、高温によって誘導されるイネの「hsp80」遺伝子と「hsp72」遺 伝子のプロモーター（Van Breusegem et al, Planta, 1994, 193, 57.)、乾燥によって 誘導されるシロイヌナズナの「rabl6」遺伝子のプロモーター（Nundy et al., Proc. Natl . Acad. Sci. USA, 1990, 87, 1406.)、紫外線の照射によって誘導されるパセリのカル コン合成酵素遺伝子のプロモーター（Schulze-Lefert et al., EMBO J., 1989, 8, 651. )、嫌気的条件で誘導されるトウモロコシのアルコールデヒドロゲナーゼ遺伝子のプロ モーター（Walker et al" Proc. Natl. Acad. Sci. USA, 1987, 84, 6624.)などが挙げら れる。また、イネキチナーゼ遺伝子のプロモーターとタバコの PRタンパク質遺伝子の プロモーターはサリチル酸などの特定の化合物によって、「rabl6」は植物ホルモンの アブシジン酸の散布によっても誘導される。 [0132] Examples of promoters for constitutive expression include the cauliflower mosaic virus 35S promoter (Odell et al., Nature, 1985, 313, 810.) and the rice actin promoter (Zhang et al., Plant). Cell, 1991, 3, 1155.) and corn ubiquitin promoter (Cornejo et al., Plant Mol. Biol, 1993, 23, 567.). In addition, promoters for inducible expression are expressed by external factors such as invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, UV irradiation, and spraying of specific compounds. There are known promoters. Such promoters include, for example, the rice chitinase gene promoter (Xu et al., Plant Mol. Biol., 1996, 30, 387.) and tapa PR protein gene promoters (Ohshima et al., Plant Cell, 1990, 2, 95.), rice-derived “lipl9” gene promoter (Aguan et al., Mol. Gene Genet., 1993) , 240,1.), Promoters of rice “hsp80” and “hsp72” genes induced by high temperature (Van Breusegem et al, Planta, 1994, 193, 57.), Arabidopsis thaliana induced by desiccation Promoter of “rabl6” gene (Nundy et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 1406.), promoter of parsley chalcone synthase gene induced by UV irradiation (Schulze-Lefert et al., EMBO J., 1989, 8, 651.), induced under anaerobic conditions. Corn alcohol dehydrogenase promoter (Walker et al "Proc. Natl. Acad. Sci. USA, 1987, 84, 6624.) In addition, the rice chitinase gene promoter and tobacco PR protein gene The promoter of certain compounds such as salicylic acid, and “ ra bl6” is a plant hormone It is also induced by application of abscisic acid.
[0133] また、本発明の DNAの導入により形質転換した細胞を効率的に選択するために、 上記組み換えベクターは、適当な選抜マーカー遺伝子を含む、もしくは選抜マーカ 一遺伝子を含むプラスミドベクターと共に細胞へ導入するのが好ましい。この目的に 使用する選抜マーカー遺伝子は、例えば抗生物質ハイグロマイシンに耐性であるハ ィグロマイシンホスホトランスフェラーゼ遺伝子、カナマイシンまたはゲンタマイシンに 耐性であるネオマイシンホスホトランスフェラーゼ、および除草剤ホスフィノスリシンに 耐性であるァセチルトランスフェラーゼ遺伝子等が挙げられる。  [0133] Further, in order to efficiently select cells transformed by introduction of the DNA of the present invention, the above recombinant vector contains an appropriate selection marker gene, or a plasmid vector containing a selection marker gene and the cell. It is preferable to introduce. Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene that is resistant to the antibiotic hygromycin, the neomycin phosphotransferase that is resistant to kanamycin or gentamicin, and the gene that is resistant to the herbicide phosphinothricin. Examples include cetyltransferase gene.
[0134] また、本発明は、本発明のベクターが導入された形質転換植物細胞を提供する。 [0134] The present invention also provides a transformed plant cell into which the vector of the present invention has been introduced.
本発明のベクターが導入される細胞としては特に制限はなぐ例えば、イネ、トウモロ コシ、コムギ、ォォムギ、ライムギ、ジャガイモ、タバコ、サトウダイコン、サトウキビ、ナタ ネ、ダイズ、ヒマヮリ、ヮタ、オレンジ、ブドウ、モモ、ナシ、リンゴ、トマト、ハクサイ、キヤ べッ、ダイコン、ニンジン、カボチヤ、キユウリ、メロン、パセリ、ラン、キク、ユリ、サフラ ン、などの植物の細胞が挙げられる力 ユーカリ、マツ、ァカシャ、ポプラ、スギ、ヒノ キ、タケ、ィチイなどの樹木が望ましい。また、本発明の植物細胞には、培養細胞の 他、植物体中の細胞も含まれる。また、プロトプラスト、苗条原基、多芽体、毛状根も 含まれる。  The cells into which the vector of the present invention is introduced are not particularly limited, for example, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower, ivy, orange, grape , Peach, pear, apple, tomato, cabbage, cabbage, radish, carrot, cabotya, cucumber, melon, parsley, orchid, chrysanthemum, lily, safran, etc. Trees such as poplar, cedar, cypress, bamboo, and yew are desirable. In addition to cultured cells, the plant cells of the present invention include cells in plant bodies. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots.
[0135] 上記発現ベクターを宿主植物細胞中に導入するために、さまざまな手法を用いるこ とができる。これらの手法には、形質転換因子としてァグロバタテリゥム'ッメファシェ ンス (Agrobacterium tumefaciens)または、ァグロバタテリゥム ·リゾゲネス (Agrobacteriu m rhizogenes)を用いた T- DNAによる植物細胞の形質転換方法のほかに、プロトプラ ストへの直接導入 (プロトプラストに電気パルス処理して DNAを植物細胞へ導入する エレクト口ポレーシヨン法や、リボソームなどとプロトプラストとの融合法、マイクロインジ ェクシヨン法、ポリエチレングリコール法など)、パーティクルガン法などが挙げられる。  [0135] In order to introduce the expression vector into a host plant cell, various techniques can be used. These methods include methods for transforming plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transforming factors. In addition, direct introduction into protoplasts (elect mouth method that introduces DNA into plant cells by electropulsing the protoplast, fusion method of ribosome and protoplast, microinjection method, polyethylene glycol method, etc.), Examples include the particle gun method.
[0136] また、植物ウィルスをベクターとして利用することによって、 目的遺伝子を植物体に 導入することができる。利用可能な植物ウィルスとしては、例えば、カリフラワーモザィ クウィルスが挙げられる。すなわち、まず、ウィルスゲノムを大腸菌由来のベクターな どに挿入して組換え体を調製した後、ウィルスのゲノム中に、これらの目的遺伝子を 挿入する。このようにして修飾されたウィルスゲノムを制限酵素によって該組換え体か ら切り出し、植物体に接種することによって、これらの目的遺伝子を植物体に導入す ることがでさる (Hohnら (1982)、 Molecular Biology of Plant Tumors (Academic Press ^ New York) pp549、米国特許第 4,407,956号明細書）。植物細胞や植物体へのベクタ 一導入の手法は、これらのみに限定されず、その他の可能性も含まれる。 [0136] In addition, a target gene can be introduced into a plant by using a plant virus as a vector. Examples of plant viruses that can be used include cauliflower mosaic virus. That is, first, after preparing the recombinant by inserting the viral genome into a vector derived from E. coli, these target genes are inserted into the viral genome. insert. It is possible to introduce these target genes into a plant by excising the viral genome thus modified from the recombinant with a restriction enzyme and inoculating the plant (Hohn et al. (1982) Molecular Biology of Plant Tumors (Academic Press ^ New York) pp549, US Pat. No. 4,407,956). Vectors introduced into plant cells and plants are not limited to these, and include other possibilities.
[0137] プロトプラストへの直接導入では、必要とされるベクターに制限はない。例えば、 pU C誘導体のような単純なプラスミドを用いることができる。 目的の遺伝子を植物細胞に 導入する方法によっては、他の DNA配列が必要になることもある。例えば、 Ήまたは R iプラスミドを植物細胞の形質転換に用いる場合には、 Tiおよび Riプラスミドの T-DNA 領域の少なくとも右の端の配列、大抵は両側の端の配列を、導入されるべき遺伝子 の隣接領域となるように接続しなければならな 、。  [0137] For direct introduction into protoplasts, there are no restrictions on the required vectors. For example, a simple plasmid such as a pUC derivative can be used. Depending on how the gene of interest is introduced into the plant cell, other DNA sequences may be required. For example, when Ή or Ri plasmids are used for transformation of plant cells, the sequence of at least the right end of the T-DNA region of Ti and Ri plasmids, usually the sequences at both ends, must be inserted into the gene to be introduced. Must be connected to be adjacent to the.
[0138] ァグロバクテリウム属菌を形質転換に用いる場合には、導入すべき遺伝子を、特別 のプラスミド、すなわち中間ベクターまたはバイナリーベクターの中にクロー-ングす る必要がある。中間ベクターはァグロバクテリウム属菌の中では複製されない。中間 ベクターは、ヘルパープラスミドあるいはエレクト口ポレーシヨンによってァグロバタテリ ゥム属菌の中に移行される。中間ベクターは、 T-DNAの配列と相同な領域をもった め、相同的組換えによって、ァグロバクテリウム属菌の Ήまたは Riプラスミド中に取り込 まれる。宿主として使われるァグロバクテリウム属菌には、 vir領域が含まれている必要 がある。通常 Ήまたは Riプラスミドに vir領域が含まれており、その働きにより、 T-DNA を植物細胞に移行させることができる。  [0138] When Agrobacterium is used for transformation, the gene to be introduced needs to be cloned into a special plasmid, that is, an intermediate vector or a binary vector. Intermediate vectors are not replicated in Agrobacterium. The intermediate vector is transferred into the genus Agrobataterium by a helper plasmid or electoral position. The intermediate vector has a region that is homologous to the T-DNA sequence, and is incorporated into the Agrobacterium sputum or Ri plasmid by homologous recombination. Agrobacterium used as a host must contain the vir region. Usually, vir or Ri plasmid contains vir region, and T-DNA can be transferred to plant cells by its function.
[0139] 一方、バイナリーベクターはァグロバクテリウム属菌の中で複製、維持され得るので 、ヘルパープラスミドあるいはエレクト口ポレーシヨン法によってァグロバタテリゥム属 菌中に取り込まれると、宿主の vir領域の働きによって、バイナリーベクター上の T-DN Aを植物細胞に移行させることができる。  [0139] On the other hand, a binary vector can be replicated and maintained in an Agrobacterium, so when incorporated into an Agrobacterium by the helper plasmid or the electroporation method, the vir region of the host By function, T-DN A on the binary vector can be transferred to plant cells.
[0140] なお、このようにして得られた中間ベクターまたはバイナリーベクター、及びこれを 含む大腸菌ゃァグロバクテリウム属菌等の微生物も本発明の対象である。  [0140] It should be noted that the intermediate vector or binary vector thus obtained and microorganisms such as Escherichia coli containing the same are also objects of the present invention.
[0141] また、本発明は、上記の形質転換植物細胞から再分化した形質転換植物体、該形 質転換植物体の子孫またはクローンである形質転換植物体、および該形質転換植 物体の繁殖材料を提供する。このような形質転換植物体は、植物種によって細胞壁 成分や細胞形態形成が改変された有用な形質転換植物体である。本発明における 細胞壁成分の改変としては、特に制限されるものではなぐ例えば、高セルロース、 低リグニン、細胞壁の厚いもの、薄いもの、繊維長の長いもの、短いもの等様々な量 的かつ質的な変化が挙げられる。また、細胞形態の改変としては、細胞伸長の変化、 細胞の大きさの変化 (体積の量的変化)などが例示できるが、これらに限定されるもの ではない。 [0141] The present invention also provides a transformed plant regenerated from the transformed plant cell, a transformed plant that is a descendant or clone of the transformed plant, and the transformed plant. Provide material breeding material. Such a transformed plant is a useful transformed plant in which cell wall components and cell morphogenesis are modified by plant species. The modification of the cell wall component in the present invention is not particularly limited, for example, high cellulose, low lignin, a thick cell wall, a thin cell, a long fiber length, a short one, various quantitative and qualitative. Change. Examples of the modification of the cell morphology include, but are not limited to, changes in cell elongation and changes in cell size (quantitative change in volume).
[0142] 本発明における形質転換植物体は、例えば、植物種によって細胞壁合成量増大に よる植物の成長量の増加、繊維細胞形態の変化、農作物の有用成分の増加などの 新たな価値を有する植物として有用である。また、細胞壁合成制御による新素材の 開発、飼料作物の消化吸収効率の増加、繊維細胞形態の変化などの新たな価値を 有する植物として有用である。  [0142] The transformed plant according to the present invention has, for example, a plant having a new value such as an increase in the amount of plant growth due to an increase in the amount of cell wall synthesis, a change in fiber cell morphology, an increase in useful components of crops depending on the plant species. Useful as. It is also useful as a plant with new value, such as the development of new materials by controlling cell wall synthesis, increased digestion and absorption efficiency of forage crops, and changes in fiber cell morphology.
[0143] 本発明にお ヽて「形質転鎌物体」とは、上述した形質転鎌物細胞を有する植 物体であり、例えば、上記形質転換細胞から再生された形質転換植物体がこれに含 まれる。形質転換された植物細胞から個体を再生する方法は植物細胞の種類により 異なるが、例えばイネでは Fujimuraら（Fujimura et al, Plant Tissue Culture Lett., 2, 74, 1995)の方法、トウモロコシでは、 Shillitoら（Shillito et al., Bio/Technology, 7, 581, 1989)の方法、ジャガイモでは、 Visserら（Visser et al., Theor. Appl. Genet., 78, 589, 1989)の方法、シロイヌナズナでは Akamaらの方法（Akama et al., Plant Cell Rep., 1 2, 7, 1992)ユーカリでは土肥らの方法 (特願平 11-127025)が挙げられる。これらの方 法により作出された形質転換植物体またはその繁殖媒体 (例えば種子、塊茎、切穂 など)から得た形質転換植物体は本発明の対象である。  [0143] In the present invention, the "transformed sickle object" is a plant having the above-described transformed sickle cell, and includes, for example, a transformed plant regenerated from the transformed cell. Be turned. The method of regenerating an individual from transformed plant cells varies depending on the type of plant cell. For example, the method of Fujimura et al. (Fujimura et al, Plant Tissue Culture Lett., 2, 74, 1995) is used for rice, and Shillito is used for maize. (Shillito et al., Bio / Technology, 7, 581, 1989), in potato, in Visser et al., Theor. Appl. Genet., 78, 589, 1989, in Arabidopsis, in Akama In the eucalyptus method (Akama et al., Plant Cell Rep., 12, 7, 1992), the method of Toi et al. (Japanese Patent Application No. 11-127025) can be mentioned. Transformed plants produced by these methods or their propagation media (eg, seeds, tubers, cut ears, etc.) are the subject of the present invention.
[0144] 本発明は、植物、特に樹木の木繊維細胞壁形成に関わる遺伝子群と、或いはこれ らの相同体、或いはこれらの遺伝子に連なるプロモーター領域を有する発現べクタ 一を宿主細胞に導入して形質転換細胞を得て、該形質転換細胞から形質転換植物 体を再生し、得られた形質転換植物体から植物種子を得て、該植物種子から植物体 を生産する工程を含む。  [0144] The present invention introduces into a host cell an expression vector having a gene group involved in plant fiber cell wall formation in plants, particularly trees, or homologues thereof, or a promoter region linked to these genes. The method includes the steps of obtaining a transformed cell, regenerating a transformed plant from the transformed cell, obtaining a plant seed from the obtained transformed plant, and producing the plant from the plant seed.
[0145] 形質転,物体から植物種子を得る工程とは、例えば、形質転,物体を発根培 地から採取し、水を含んだ土を入れたポットに移植し、一定温度下で生育させて、花 を形成させ、最終的に種子を形成させる工程を指す。また、種子から植物体を生産 する工程とは、例えば、形質転換植物体上で形成された種子が成熟したところで、単 離して、水を含んだ土に播種し、一定温度、照度下で生育させることにより、植物体 を生産する工程をいう。 [0145] The process of obtaining plant seeds from the transformation and the object is, for example, the cultivation of the transformation and the object. It refers to the process of collecting from the ground, transplanting it to a pot containing water-containing soil, growing it at a constant temperature, forming flowers, and finally forming seeds. In addition, the process of producing a plant from a seed is, for example, when the seed formed on a transformed plant matures, is isolated and sown in soil containing water, and grows under a constant temperature and illuminance. This refers to the process of producing a plant body.
[0146] なお、形質転換植物体中の導入された外来 DNAまたは核酸の存在は、公知の PCR 法やサザンハイブリダィゼーシヨン法によって、または植物体中の核酸の塩基配列を 解析することによって確認することができる。この場合、形質転,物体からの DNA または核酸の抽出は、公知の J.Sambrookらの方法（Molecular Cloning,第 2版， Cold SpringHarbor laboratory Press, 1989)に準じて実施することができる。  [0146] The presence of the introduced foreign DNA or nucleic acid in the transformed plant body is determined by a known PCR method or Southern hybridization method, or by analyzing the nucleotide sequence of the nucleic acid in the plant body. Can be confirmed. In this case, transformation and extraction of DNA or nucleic acid from the object can be performed according to the known method of J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor laboratory Press, 1989).
[0147] 植物体中に存在する本発明の DNAを、 PCR法を用いて解析する場合には、上記の ように再生植物体力ゝら抽出した核酸を铸型として増幅反応を行う。また、本発明の DN Aの塩基配列に従って適当に選択された塩基配列をもつ合成したオリゴヌクレオチド をプライマーとして用い、これらを混合させた反応液中お 、て増幅反応を行うこともで きる。増幅反応においては、 DNAの変性、アニーリング、伸張反応を数十回繰り返す と、本発明の DNA配列を含む DNA断片の増幅生成物を得ることができる。増幅生成 物を含む反応液を、例えばァガロース電気泳動にかけると、増幅された各種の DNA 断片が分画されて、その DNA断片が本発明の DNAに対応することを確認することが 可能である。  [0147] When the DNA of the present invention present in a plant body is analyzed by PCR, an amplification reaction is carried out using the nucleic acid extracted from the regenerated plant body as described above in a cage shape. In addition, an amplification reaction can also be carried out in a reaction mixture in which a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of DNA of the present invention is used as a primer and these are mixed. In the amplification reaction, when DNA denaturation, annealing, and extension reactions are repeated several tens of times, an amplification product of a DNA fragment containing the DNA sequence of the present invention can be obtained. When the reaction solution containing the amplified product is subjected to, for example, agarose electrophoresis, it is possible to confirm that the amplified DNA fragments are fractionated and that the DNA fragments correspond to the DNA of the present invention. .
[0148] ー且、染色体内に本発明の DNAが導入された形質転換植物体が得られれば、該 植物体力も有性生殖または無性生殖により子孫を得ることが可能である。また、該植 物体やその子孫あるいはクローン力 繁殖材料 (例えば種子、果実、切穂、塊茎、塊 根、株、カルス、プロトプラスト等）を得て、それらを基に該植物体を量産することも可 能である。  [0148]-If a transformed plant into which the DNA of the present invention is introduced into the chromosome is obtained, it is possible to obtain offspring by sexual reproduction or asexual reproduction. It is also possible to obtain the plant, its progeny or clonal power propagation material (eg, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) and mass-produce the plant based on them. Yes, it is possible.
[0149] また本発明は、配列番号： 1〜34、 101〜111に記載の塩基配列の全部又は一部 を増幅するプライマーを提供する。本発明のプライマーは、本発明の検査方法に使 用することが可能である。本発明のプライマーは、本発明の DNAまたはその相補鎖の 少なくとも一部を増幅しうるものであれば、特に制限されるものではないが、その長さ は通常 15bp〜100bpであり、好ましくは 16bp〜31bpであり、さらに好ましくは、例えば、 16、 17、 18、 19、 20、 21、 22、 23、 24、 25、 31ヌクレオチドである。本発明のプライマー は、 3'側の領域は相補的とし、 5'側には制限酵素認識配列やタグなどを付加してもよ い。 [0149] The present invention also provides a primer that amplifies all or a part of the base sequences set forth in SEQ ID NOs: 1-34 and 101-111. The primer of the present invention can be used in the inspection method of the present invention. The primer of the present invention is not particularly limited as long as it can amplify at least a part of the DNA of the present invention or its complementary strand. Is usually 15 bp to 100 bp, preferably 16 bp to 31 bp, and more preferably, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 31 nucleotides. In the primer of the present invention, the 3 ′ region may be complementary, and a restriction enzyme recognition sequence or a tag may be added to the 5 ′ side.
さらに本発明は、配列番号： 1〜34、 101〜： L 11に記載の塩基配列の全部又は一 部を増幅するプライマーセットを提供する。具体的には、本発明のプライマーセットと しては、  Furthermore, the present invention provides a primer set for amplifying all or part of the base sequence described in SEQ ID NOs: 1-34, 101-: L11. Specifically, the primer set of the present invention includes:
(a)配列番号： 69に記載の塩基配列力 なる DNAおよび配列番号： 70に記載の塩 基配列からなる DNA (配列番号： 3に記載の塩基配列の全部または一部を検出する プライマーセット)、  (a) DNA having the nucleotide sequence described in SEQ ID NO: 69 and DNA consisting of the nucleotide sequence described in SEQ ID NO: 70 (primer set for detecting all or part of the nucleotide sequence described in SEQ ID NO: 3) ,
(b)配列番号： 71に記載の塩基配列力もなる DNAおよび配列番号： 72に記載の塩 基配列からなる DNA (配列番号: 6に記載の塩基配列の全部または一部を検出する プライマーセット)、  (b) DNA having the nucleotide sequence described in SEQ ID NO: 71 and DNA consisting of the nucleotide sequence described in SEQ ID NO: 72 (primer set for detecting all or part of the nucleotide sequence described in SEQ ID NO: 6) ,
(c)配列番号： 73に記載の塩基配列力 なる DNAおよび配列番号： 74に記載の塩 基配列からなる DNA (配列番号： 11に記載の塩基配列の全部または一部を検出する プライマーセット)、  (c) DNA having the nucleotide sequence described in SEQ ID NO: 73 and DNA consisting of the nucleotide sequence described in SEQ ID NO: 74 (primer set for detecting all or part of the nucleotide sequence described in SEQ ID NO: 11) ,
(d)配列番号： 75に記載の塩基配列力 なる DNAおよび配列番号： 76に記載の塩 基配列からなる DNA (配列番号： 14に記載の塩基配列の全部または一部を検出する プライマーセット)、  (d) DNA having the nucleotide sequence described in SEQ ID NO: 75 and DNA consisting of the nucleotide sequence described in SEQ ID NO: 76 (primer set for detecting all or part of the nucleotide sequence described in SEQ ID NO: 14) ,
(e)配列番号： 77に記載の塩基配列力 なる DNAおよび配列番号： 78に記載の塩 基配列からなる DNA (配列番号： 19に記載の塩基配列の全部または一部を検出する プライマーセット)、  (e) DNA having the nucleotide sequence described in SEQ ID NO: 77 and DNA consisting of the nucleotide sequence described in SEQ ID NO: 78 (primer set for detecting all or part of the nucleotide sequence described in SEQ ID NO: 19) ,
(f)配列番号： 79に記載の塩基配列力 なる DNAおよび配列番号: 80に記載の塩基 配列からなる DNA (配列番号： 23に記載の塩基配列の全部または一部を検出するプ ライマーセット)、  (f) DNA having the nucleotide sequence of SEQ ID NO: 79 and DNA consisting of the nucleotide sequence of SEQ ID NO: 80 (a primer set for detecting all or part of the nucleotide sequence of SEQ ID NO: 23) ,
(g)配列番号： 81に記載の塩基配列からなる DNAおよび配列番号： 82に記載の塩 基配列からなる DNA (配列番号： 24に記載の塩基配列の全部または一部を検出する プライマーセット)、 (h)配列番号: 83に記載の塩基配列力 なる DNAおよび配列番号: 84に記載の塩 基配列からなる DNA (配列番号： 25に記載の塩基配列の全部または一部を検出する プライマーセット)、 (g) DNA consisting of the base sequence set forth in SEQ ID NO: 81 and DNA consisting of the base sequence set forth in SEQ ID NO: 82 (Primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 24) , (h) DNA having the nucleotide sequence described in SEQ ID NO: 83 and DNA consisting of the nucleotide sequence described in SEQ ID NO: 84 (a primer set for detecting all or part of the nucleotide sequence described in SEQ ID NO: 25) ,
(i)配列番号： 123に記載の塩基配列力もなる DNAおよび配列番号： 124に記載の 塩基配列からなる DNA (配列番号： 101に記載の塩基配列の全部または一部を検出 するプライマーセット）、  (i) DNA having the base sequence ability described in SEQ ID NO: 123 and DNA consisting of the base sequence described in SEQ ID NO: 124 (primer set for detecting all or part of the base sequence described in SEQ ID NO: 101),
(j)配列番号： 125に記載の塩基配列力もなる DNAおよび配列番号： 126に記載の 塩基配列からなる DNA (配列番号： 102に記載の塩基配列の全部または一部を検出 するプライマーセット）、  (j) DNA having the nucleotide sequence described in SEQ ID NO: 125 and DNA consisting of the nucleotide sequence described in SEQ ID NO: 126 (primer set for detecting all or part of the nucleotide sequence described in SEQ ID NO: 102),
(k)配列番号： 127に記載の塩基配列力もなる DNAおよび配列番号： 128に記載の 塩基配列からなる DNA (配列番号： 103に記載の塩基配列の全部または一部を検出 するプライマーセット）、  (k) DNA having the nucleotide sequence ability set forth in SEQ ID NO: 127 and DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 128 (primer set for detecting all or part of the nucleotide sequence set forth in SEQ ID NO: 103),
(1)配列番号： 129に記載の塩基配列力もなる DNAおよび配列番号： 130に記載の 塩基配列からなる DNA (配列番号： 104に記載の塩基配列の全部または一部を検出 するプライマーセット）、  (1) DNA having the nucleotide sequence ability described in SEQ ID NO: 129 and DNA consisting of the nucleotide sequence described in SEQ ID NO: 130 (primer set for detecting all or part of the nucleotide sequence described in SEQ ID NO: 104),
(m)配列番号： 131に記載の塩基配列からなる DNAおよび配列番号： 132に記載の 塩基配列からなる DNA (配列番号： 105に記載の塩基配列の全部または一部を検出 するプライマーセット）、  (m) DNA consisting of the base sequence set forth in SEQ ID NO: 131 and DNA consisting of the base sequence set forth in SEQ ID NO: 132 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 105),
(n)配列番号： 133に記載の塩基配列力もなる DNAおよび配列番号： 134に記載の 塩基配列からなる DNA (配列番号： 106に記載の塩基配列の全部または一部を検出 するプライマーセット）、  (n) DNA having the nucleotide sequence described in SEQ ID NO: 133 and DNA consisting of the nucleotide sequence described in SEQ ID NO: 134 (primer set for detecting all or part of the nucleotide sequence described in SEQ ID NO: 106),
(o)配列番号： 135に記載の塩基配列からなる DNAおよび配列番号： 136に記載の 塩基配列からなる DNA (配列番号： 107に記載の塩基配列の全部または一部を検出 するプライマーセット）、  (o) DNA consisting of the base sequence set forth in SEQ ID NO: 135 and DNA consisting of the base sequence set forth in SEQ ID NO: 136 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 107),
(P)配列番号： 137に記載の塩基配列からなる DNAおよび配列番号： 138に記載の 塩基配列からなる DNA (配列番号： 108に記載の塩基配列の全部または一部を検出 するプライマーセット）、  (P) DNA consisting of the base sequence set forth in SEQ ID NO: 137 and DNA consisting of the base sequence set forth in SEQ ID NO: 138 (a primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 108),
(q)配列番号： 139に記載の塩基配列からなる DNAおよび配列番号： 140に記載の 塩基配列からなる DNA (配列番号： 109に記載の塩基配列の全部または一部を検出 するプライマーセット）、 (q) DNA consisting of the base sequence described in SEQ ID NO: 139 and SEQ ID NO: 140 DNA consisting of a base sequence (a primer set for detecting all or part of the base sequence described in SEQ ID NO: 109),
(r)配列番号： 141に記載の塩基配列力 なる DNAおよび配列番号： 142に記載の 塩基配列からなる DNA (配列番号： 110に記載の塩基配列の全部または一部を検出 するプライマーセット）、  (r) DNA having the nucleotide sequence of SEQ ID NO: 141 and DNA consisting of the nucleotide sequence of SEQ ID NO: 142 (primer set for detecting all or part of the nucleotide sequence of SEQ ID NO: 110),
(s)配列番号： 143に記載の塩基配列力 なる DNAおよび配列番号： 144に記載の 塩基配列からなる DNA (配列番号：111に記載の塩基配列の全部または一部を検出 するプライマーセット）、  (s) DNA having the nucleotide sequence described in SEQ ID NO: 143 and DNA consisting of the nucleotide sequence described in SEQ ID NO: 144 (primer set for detecting all or part of the nucleotide sequence described in SEQ ID NO: 111),
を例示することが出来る。  Can be illustrated.
[0151] また上記のプライマー以外にも、当業者であれば、配列番号： 1〜34、 101〜： L 11 に記載の塩基配列を利用して、同様の機能を有するプライマーセットを作成すること が可能である。本発明のプライマーには、このようなプライマーもまた含まれる。本発 明の PCRプライマーは、当業者においては、例えば、自動オリゴヌクレオチド合成機 等を利用して作製することができる。  [0151] In addition to the primers described above, those skilled in the art should create a primer set having the same function by using the base sequences described in SEQ ID NOs: 1-34, 101-: L11. Is possible. Such primers are also included in the primer of the present invention. The PCR primer of the present invention can be prepared by those skilled in the art using, for example, an automatic oligonucleotide synthesizer.
[0152] 本発明はまた、上記プライマーセットを含む試薬を提供する。本発明の試薬は、本 発明の検査方法に使用することが可能である。このような試薬には、上記に記載の検 查方法に使用されるものを含みうる。例えば、本発明の遺伝子、プライマー、染色液 等を挙げることができる。その他、蒸留水、塩、緩衝液、タンパク質安定剤、保存剤等 が含まれていてもよい。  [0152] The present invention also provides a reagent comprising the above primer set. The reagent of the present invention can be used in the inspection method of the present invention. Such reagents may include those used in the above-described detection methods. For example, the gene, primer, staining solution and the like of the present invention can be mentioned. In addition, distilled water, salt, buffer solution, protein stabilizer, preservative and the like may be contained.
[0153] 本発明における形質転換植物体をクローン植林により大規模に育成することによつ て、セルロースを核にしたバイオマスの安定的供給が可能となる。現在、化石資源は 工業生産において、原材料や燃料 (エネルギー）として大量に使われている。特にェ ネルギー代替にっ 、ては、木質バイオマスを直接燃焼する (薪利用）ことが発展途上 国で日常的に行われている力 これをさらに利用しやすい形態（アルコール：ェチル アルコール）に変換することにより有効な対応が可能であると考えられる。実際、エタ ノールはブラジルなどでサトウキビの絞り汁等に由来する廃糖蜜力 アルコール発酵 により生産され、自動車燃料として用いられており、アメリカ、 EUにおいてもサッマイ モゃトウモロコシのデンプンをいったん加水分解させブドウ糖にしてからアルコール 発酵を行っている事例が増えている。アメリカでは 1999年 8月、「2010年までにバイオ マスのエネルギー活用を一次エネルギー全体の 10%に高める」と発表し、その目的 のひとつ力 バイオマスカも精製したエタノールをガソリンに混ぜて使う方法である。 具体的事例としては、トウモロコシを原料とする gasohol (ガソリンへのエタノールのブ レンド割合が 10%)というエタノール燃料であり、コーンベルト地帯を中心とする 20州 で使用され、米国の自動車燃料全体の約 1%を占めており、砂糖キビの採れる特定 の州ではガソリンの 40%のシェアを占めており、アメリカ国内自動車全メーカーが gaso hoiを燃料として保証し、具体的にはゼネラルモーター社やクライスラー社はこの利用 を推奨して 、る。 EUでは 2010年に 1990年レベルの 8%の温室効果ガス削減をめざし 、再生可能エネルギーのシェアを全エネルギーの 12%にする計画が進行しており、 自動車代替燃料にっ 、ては、 2005年までに化石燃料の 5%をバイオ燃料 (生物資源 由来の燃料)で代替するという目標を設定している。 EUのエネルギー利用計画をみ ると、太陽電池 (貢献度 1%)、風力発電（同 19%)、バイオマス'コージ ネ（同 80%) となっており、バイオマスに大きな期待が寄せられている。また、 2015年にはエネルギ 一用のバイオ作物の栽培力 土地の最大利用面積を占める見込みとなっている。し かしながら、一方では食料増産の観点から、今後穀類やィモ類を大量に工業原料向 けに消費することには限界がある。従って、本発明により例えば、セルロース含量が 高いユーカリを大量に育成できれば、これから得られるリグノセルロースを原料に、酸 加水分解あるいは酵素分解 (セルラーゼ）工程によりグルコースを得、次いでアルコ ール発酵によりエタノールを大量に生産することが可能であり、このような工程の基礎 技術は既に確立されている。さら〖こ、グルコースを原料に生分解性プラスチック (ポリ 乳酸)を生産する技術は既に確立されており、ィモ類のデンプンを用いて工業生産 規模での実用化が進展している。し力しながら、将来的には食糧である穀類に替わり 、榭木由来のバイオマスが主流になることが予想される。尚、リグニンについても、実 用化に関しては今後技術的な課題を克服する必要があるものの、プラスチックや接 着剤などの用途が見込まれている。また、エネルギーの観点力も述べると、リグニンは 製紙産業のパルプ製造工程でィヒ学的に分解され廃液 (黒液と呼ばれる）中に含まれ るが、廃液中より必要な薬品を抽出した後に、工場内の燃料として活用しており、別 の言 、方をすると既に燃料の一部を木質バイオマスに依存して 、ることに他ならな ヽ [0153] By growing the transformed plant in the present invention on a large scale by clonal plantation, a stable supply of biomass using cellulose as a core can be achieved. At present, fossil resources are used in large quantities as raw materials and fuel (energy) in industrial production. In particular, as an alternative to energy, directly burning woody biomass (using soot) is a daily activity in developing countries. Converting this to a more accessible form (alcohol: ethyl alcohol) It is thought that effective correspondence is possible. In fact, ethanol is produced from sugarcane juice in Brazil and other countries by molasses alcohol fermentation and used as an automobile fuel. In the United States and the EU, starch of sweet potato maize is once hydrolyzed to give glucose. Alcohol Increasing cases of fermenting. In the United States in August 1999, the company announced that “by 2010, the energy use of biomass will be increased to 10% of the total primary energy.” One of its aims is to use biomass ethanol mixed with gasoline. is there. A specific example is an ethanol fuel called corn-based gasohol (10% ethanol blended to gasoline), which is used in 20 states mainly in the corn belt, It accounts for about 1%, and in certain states where sugar millet is used, it has a 40% share of gasoline, and all US car manufacturers guarantee gaso hoi as fuel, specifically General Motors and Chrysler. The company recommends this use. In 2010, the EU is planning to reduce the share of renewable energy to 12% of total energy with the aim of reducing greenhouse gas emissions by 10% in 1990 levels. By the end, the goal of substituting 5% of fossil fuel with biofuel (fuel derived from biological resources) has been set. Looking at the EU energy use plan, solar cells (contribution 1%), wind power generation (19%), and biomass co-generation (80%) have high expectations for biomass. . In 2015, it is expected that it will occupy the largest area of land used to cultivate bio-crop for energy use. However, on the other hand, from the viewpoint of increasing food production, there is a limit to consuming large quantities of cereals and potatoes for industrial raw materials in the future. Therefore, for example, if eucalyptus having a high cellulose content can be grown in large quantities according to the present invention, glucose is obtained from the lignocellulose obtained therefrom by an acid hydrolysis or enzymatic decomposition (cellulase) process, and then ethanol is obtained by alcohol fermentation. Large-scale production is possible, and the basic technology for such a process has already been established. Furthermore, the technology for producing biodegradable plastics (polylactic acid) using glucose as a raw material has already been established, and its practical application on the industrial production scale using the starch of potatoes is progressing. However, in the future, biomass derived from wood is expected to become the mainstream instead of cereal as food. Lignin is also expected to be used for plastics and adhesives, although it will be necessary to overcome technical challenges in the future. In terms of energy, lignin is decomposed ichiologically in the pulp manufacturing process of the paper industry and is contained in the waste liquid (called black liquor). After extracting the necessary chemicals from the waste liquid, It is used as fuel in the factory. In other words, it is nothing else but to rely on woody biomass for a part of the fuel.
[0154] 本発明において木質バイオマスを安定的にかつ大規模に育成し、植林による循環 を行うことによって、従来の原料としての活用に加え、バイオマス変換による石油代替 エネルギー、さらにはセルロースあるいはへミセルロースから新規なプラスチックを創 出する（こちらも技術的には可能)ことも充分考えられる。さらに、木質バイオマスの普 及は、エネルギーセキュリティや環境問題の解決策になると同時に、農林業を始め新 たな産業の開発や就労機会の創出にもつながる。 [0154] In the present invention, woody biomass is cultivated stably and on a large scale and circulated by afforestation, so that it can be used as a conventional raw material, as well as petroleum alternative energy by biomass conversion, and also cellulose or hemicellulose It is quite possible to create a new plastic from this (which is also technically possible). Furthermore, the spread of woody biomass will become a solution to energy security and environmental problems, and at the same time will lead to the development of new industries such as agriculture and forestry and the creation of employment opportunities.
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に 組み入れられる。  All prior art documents cited in the present specification are incorporated herein by reference.
実施例  Example
[0155] 以下実施例により本発明をさらに詳しく説明するが、本発明は以下の実施例に限 定されるものではない。なお、実験手法に関しては、特に記載のない限り、「クロー- ングとシークェンス」（渡辺格監修、杉浦昌弘編集、農村文化社 (1989年)）や、「Mole cularし loning (Sambrookら (1989), し old Spring Harbor Laboratory Press)」などの 験書に従った。  [0155] The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the following examples. Regarding experimental methods, unless otherwise stated, “Cloning and Sequence” (supervised by Watanabe, edited by Masahiro Sugiura, Rural Bunkasha (1989)), “Mole cular loning (Sambrook et al. (1989) , And old Spring Harbor Laboratory Press).
[0156] (実施例 1)ユーカリ ESTデータベースの作製  [0156] (Example 1) Preparation of eucalyptus EST database
(1)ユーカリからの RNA抽出  (1) RNA extraction from Eucalyptus
抽出の対象となるユーカリ組織につ!、ては、幹の肥大成長部（2次壁肥厚帯；形成 層に富む組織)、葉、根とし、傾斜刺激、塩溶液暴露によるストレス付与等様々な状 況からの遺伝子発現を想定している。抽出の基本的な方法は、日尾野らの方法 (特 願平 6— 219187)に記載の方法である。下記にはその実施例として、水耕栽培による ユーカリの根を材料に用いた RNAの抽出方法を詳細に説明する。  For eucalyptus tissue to be extracted! For example, stem growth (secondary wall thickening zone; tissue rich in formation layer), leaves, roots, slant stimulation, stress application by salt solution exposure, etc. The gene expression from the situation is assumed. The basic method of extraction is the method described by Hino et al. (Japanese Patent Application No. 6-219187). In the following, as an example, an RNA extraction method using eucalyptus roots by hydroponics as a material will be described in detail.
[0157] 2ヶ月間生育したユーカリ（Eucalyptus camaldulensis)幼植物体を水耕培養槽に移 植した。水耕栽培は Hoagland-Arnonらの培養液を用いて行った。水耕培養液の組 成は、 5.0mM KNO、 3.0mM Ca(NO )、 2.0 mM NH H PO、 2.0 mM MgSO、 47 M  [0157] Eucalyptus (Eucalyptus camaldulensis) seedlings grown for 2 months were transferred to a hydroponic culture tank. Hydroponics was performed using the culture solution of Hoagland-Arnon et al. Hydroponic cultures consisted of 5.0 mM KNO, 3.0 mM Ca (NO), 2.0 mM NH H PO, 2.0 mM MgSO, 47 M
3 3 2 4 2 4 4  3 3 2 4 2 4 4
H BO、 M MnCl、 36 M FeSO、 3.1 M ZnSO、 0.16 μ M CuSO、 75nM (NH H BO, M MnCl, 36 M FeSO, 3.1 M ZnSO, 0.16 μM CuSO, 75nM (NH
3 3 2 4 4 43 3 2 4 4 4
)6Mo O であり、脱塩水を用いて調製し、 pHは毎日 0.1Mの NaOHあるいは KOHで 6. 0に調整した。更に、 1週間毎に培養液すベてを交換した。ストレス処理を行う場合は 、この培養液に栽培 1日目以降 4日目まで最終濃度が順次 50,100,200,300mMになる ように NaClを添カ卩した培養液をストレス処理区、 NaCl無添加培養液をコントロールとし た。 4日目に根 10gを小片にし、液体窒素中で磨砕した。これを 50ml遠心チューブ (N UNC社製）に移し、ガラスビーズを 10g加えた後、ホモジナイザーで 5分間摩砕した。 これにジチオスレィトール（lmg/ml)を含むメタノール溶液を用いて上清に着色が認 められなくなるまで (3回程度)摩砕試料の溶媒抽出を繰り返した。抽出終了後、試料 を凍結乾燥させた。この凍結乾燥試料を 25mlの pH 9 lOOmM CHES緩衝液 (使用直 前に 20ミリグラムのジチオスレィトール、 10mMバナジルリボヌクレオシド化合物溶液を 添加）と混合し 65°Cで 30分間保温した。保温終了後の試料溶液中に、 5M塩化ナトリ ゥム溶液及び 10%CTAB溶液を添加した。この際、添加後の試料溶液中における塩化 ナトリウム濃度は 1.4M、 CTAB濃度は l% (w/v)となるようにした。試料溶液をよく混合 した後 65°Cで 10分間保温した後、等量のクロ口ホルム:イソアミルアルコール（ = 24： 1)溶液を添加し、緩やかに、かつ充分に混合した。混合後、遠心操作により上清を 回収した。上清に対し、 55%容のイソプロパノールを添加し、 1時間氷冷した。遠心操 作により沈殿を得、これを水に溶解させた後、フエノール抽出を行った。フエノール抽 出後の上清に 10%容の 3M酢酸ナトリウム溶液および 60%容のイソプロパノールを添カロ し、よく混合した後、遠心操作により沈殿を回収した。沈殿を滅菌水にて溶解した後、 終濃度 3Mとなるように 12M塩化リチウム溶液を添加し、よく混合した後、 1時間氷冷し た。遠心操作により RNA沈殿を回収し、洗浄、乾燥を経て、最終的に水 100 Lに溶 解させ、 totalRNA画分を得た。この結果、ストレス処理区の根、コントロールの根から 、それぞれ 610 gの全 RNAを得ることが出来た。全 RNA画分からの mRNAの精製は P olyATtract mRNA Isolation Systemlll &IVキット（Cat.# Z5300& Z5310プロメガ社製 USA)を用いた。この結果、 610 gの totalRNAからストレス処理区サンプルからは 1.3 μ g、コントロールサンプルからは 1.8 μ gの mRNAを得た。 6Mo 2 O, prepared with demineralized water, pH is 0.1M NaOH or KOH daily 6. Adjusted to 0. Furthermore, all the culture medium was changed every week. When stress treatment is performed, the culture solution supplemented with NaCl so that the final concentration is 50,100,200,300 mM in order from the first day to the fourth day of cultivation is controlled in the stress treatment area, and the culture solution without addition of NaCl is controlled. And On the fourth day, 10 g of the roots were cut into small pieces and ground in liquid nitrogen. This was transferred to a 50 ml centrifuge tube (manufactured by NUNC), 10 g of glass beads were added, and then ground for 5 minutes with a homogenizer. A methanol solution containing dithiothreitol (lmg / ml) was used for this, and solvent extraction of the ground sample was repeated until no coloration was observed in the supernatant (about 3 times). After extraction, the sample was lyophilized. This lyophilized sample was mixed with 25 ml of pH 9 lOOmM CHES buffer (20 milligrams of dithiothreitol and 10 mM vanadyl ribonucleoside compound solution added immediately before use) and incubated at 65 ° C for 30 minutes. A 5M sodium chloride solution and a 10% CTAB solution were added to the sample solution after the incubation. At this time, the sodium chloride concentration in the sample solution after addition was 1.4 M, and the CTAB concentration was 1% (w / v). The sample solution was mixed well and kept at 65 ° C. for 10 minutes, and then an equal amount of Kuroguchi form: isoamyl alcohol (= 24: 1) solution was added and gently and thoroughly mixed. After mixing, the supernatant was collected by centrifugation. To the supernatant, 55% volume of isopropanol was added and ice-cooled for 1 hour. A precipitate was obtained by centrifugation and dissolved in water, followed by phenol extraction. The supernatant after phenol extraction was supplemented with 10% 3M sodium acetate solution and 60% isopropanol, mixed well, and the precipitate was collected by centrifugation. After the precipitate was dissolved in sterilized water, 12M lithium chloride solution was added to a final concentration of 3M, mixed well, and then ice-cooled for 1 hour. The RNA precipitate was collected by centrifugation, washed and dried, and finally dissolved in 100 L of water to obtain a total RNA fraction. As a result, 610 g of total RNA was obtained from each of the roots of the stress-treated group and the control root. For purification of mRNA from the total RNA fraction, PolyATtract mRNA Isolation Systemlll & IV kit (Cat. # Z5300 & Z5310 Promega Corp. USA) was used. As a result, 1.3 μg of the mRNA was obtained from 610 g of total RNA and 1.8 μg of mRNA was obtained from the control sample.
(2) cDNAライブラリーの構築 (2) Construction of cDNA library
(1)で示した方法により得られた各種組織、状況由来のユーカリ mRNAを、クロンテ ック社の Smart cDNA library construction kitを用いて cDNAを合成し、最終的にファ ージミドライブラリーとした。こうして作成された cDNAライブラリ一は、各々カ^ X 106pfo 以上の独立のクローン力もなるものであった。尚、ライブラリーの増幅は作製した一部 に対して行 、、クローンの解析には増幅をかけずに供した。 The eucalyptus mRNA derived from various tissues and situations obtained by the method shown in (1) was synthesized using the Clontech Smart cDNA library construction kit, and finally the eucalyptus mRNA was synthesized. -Dimid library. Each cDNA library created in this way also had independent clone power of more than 10 X 10 6 pfo. Library amplification was performed on a part of the library prepared, and the clone was analyzed without amplification.
[0159] (3) cDNAクローンの解読とデータベース構築 [0159] (3) Decoding cDNA clones and database construction
各組織由来のユーカリファージミド cDNAライブラリーより、クローンをランダムに選抜 し、プラスミドを精製後、アマシャム社のダイターミネータ一シークェンスキットによる酵 素反応を経て、同社の大規模高速シーケンサーを用いて、塩基配列データの取得を 行った。  Randomly select clones from a eucalyptus phagemid cDNA library derived from each tissue, purify the plasmid, undergo an enzyme reaction with Amersham's dye terminator sequence kit, and use the company's large-scale high-speed sequencer to base sequence Data was acquired.
データ解析は、プラスミド由来の既知配列を削除した後、解析ソフトウェアにより、ク ラスタリング操作により相同配列の抽出を行った。その後、遺伝子情報データベース の 1つである米国 ' GenBankの全データベースとの比較検索により、おおよその機能 予測（ァノテーシヨン）をおこなった。  For data analysis, after deleting a known sequence derived from a plasmid, homologous sequences were extracted by a clustering operation using analysis software. After that, an approximate function prediction (annotation) was made by comparison search with all databases of US GenBank, one of the gene information databases.
[0160] (実施例 2)細胞壁形成への関与が推定される遺伝子群の選抜 [0160] (Example 2) Selection of genes that are presumed to be involved in cell wall formation
得られたァノテーシヨン情報を用いて、セルロースやリグニン合成に関わる遺伝子（ Proc Natl Acad Sci U S A 2001 Dec 4;98(25): 14732-7.,特開 2000- 41685、特開 200 2-95482、特開 2003-180372)と高い相同性を持つクローンの検索を行った。その結 果、 59クローンを特定した。し力しながら、これらのクローンの中には木繊維形成だけ でなぐ榭皮組織等の形成に関わるものや細胞壁合成とは関係な 、ものも含まれて いる。  Using the obtained annotation information, genes related to cellulose and lignin synthesis (Proc Natl Acad Sci USA 2001 Dec 4; 98 (25): 14732-7., JP 2000-41685, JP 2002-95482, JP We searched for clones with high homology. As a result, 59 clones were identified. However, some of these clones include those involved in the formation of scab tissue and the like that are formed only by wood fiber formation and those that are not related to cell wall synthesis.
[0161] (実施例 3)木繊維細胞壁形成に関わる遺伝子群の抽出  [0161] (Example 3) Extraction of genes involved in wood fiber cell wall formation
(1)ユーカリ榭幹特異的オリゴマイクロアレイの作製  (1) Preparation of Eucalyptus trunk specific oligo microarray
上述のユーカリ ESTデータベースより、ユーカリオリゴマイクロアレイの作製を行った 。なお、実際の作製は、アジレントテクノロジ一社（日本国内代理店は、横河アナリテ ィカルシステムズ）に委託した。詳細については下記ホームページに記述されている o http： // www. chem . agilent · com/cag/ country/ JP/ products/PL, ol494.htm  A eucalyptus oligo microarray was prepared from the eucalyptus EST database. The actual production was outsourced to Agilent Technologies, Inc. (Yokogawa Analytical Systems is the Japanese distributor). Details are described on the following website: o http: // www. Chem. Agilent · com / cag / country / JP / products / PL, ol494.htm
このようにして作製したユーカリオリゴマイクロアレイは、 8400個のオリゴ DNAを収録 しており、ユーカリ榭幹部で発現を認める遺伝子の大部分を網羅可能である。  The eucalyptus oligo microarray produced in this way contains 8400 oligo DNAs, and can cover most of the genes that are expressed in eucalyptus trunks.
[0162] (2)マイクロアレイ解析によるユーカリ榭幹の木繊維細胞壁形成に関わる遺伝子群の 抽出 [0162] (2) Gene array related to eucalyptus trunk tree fiber cell wall formation by microarray analysis Extraction
榭幹における木質バイオマスの大部分は木繊維と呼ばれる組織細胞で構成されて いる。この木繊維の性質を決める主要要素として、木繊維組織の比重'細胞壁構造 や成分 (セルロース'へミセルロース 'リグニン)等がある。これらは、単一の遺伝子や タンパク質で規定されるものではなぐ多数の遺伝子が群となり協調して働くことで形 成される。一方、木繊維の性質は、広葉樹 '針葉樹を問わず、地上高'種により変動 することが知られている。このような材料に対し、 ESTデータ解析とマイクロアレイによ る遺伝子発現解析を行うことで、木繊維形成時にぉ 、て協調して働く一連の遺伝子 を総括的に捉えることが可能となり、各構成遺伝子の発現状態を捉えることも可能で ある。  Most of the woody biomass in the trunk is composed of tissue cells called wood fibers. The main factors that determine the properties of wood fibers include the specific gravity of the wood fiber structure, the cell wall structure and components (cellulose, hemicellulose, lignin). These are formed by working together in a group of many genes that are not defined by a single gene or protein. On the other hand, the properties of wood fibers are known to vary depending on the species of hardwood 'coniferous trees, high above ground'. By performing EST data analysis and microarray gene expression analysis on such materials, it becomes possible to comprehensively capture a series of genes that work together in the formation of wood fibers. It is also possible to capture the expression state of.
[0163] ァノテーシヨン情報をもとに選抜された 59クローンの中には、木繊維形成だけでなく 、榭皮組織等の形成に関わるものや細胞壁合成とは関係な 、ものも含まれて 、る。 そこで、異なる地上高や種のユーカリ榭幹を用いて、マイクロアレイによる発現解析に より、これらの中から木繊維細胞壁形成に関わる遺伝子群を抽出した。  [0163] Among the 59 clones selected on the basis of annotation information, not only wood fiber formation but also those involved in the formation of crusted tissue, etc., and those related to cell wall synthesis are included. . Therefore, genes related to the formation of wood fiber cell walls were extracted from these by eucalyptus trunks of different ground heights and species, by microarray expression analysis.
[0164] 本発明者らは、実施例 1に示した RNA抽出方法に従って、 5年生のカマルドレンシ ス種の樹幹基部と中央部、グロプルス種を材料に全 RNAを抽出した。この際、各植物 体の榭皮側（師部）と木繊維形成組織側 (木部)を用いた。各個体から得られた全 RN A力 、プロメガ社製の PolyATract mRNA Isolation Systemにより、 mRNAを精製した。 このようにして得られた合計 4種の mRNAを铸型に合成した各 cRNAを、各々 2種の蛍 光色素（cy3.cy5)により標識し、オリゴマイクロアレイ解析におけるプローブとして用い 、ノ、イブリダィゼーシヨンを行った（図 1〜3)。標識を含むハイブリダィゼーシヨンの方 法はアジレントテクノロジーより提示の解析プロトコルに従った。スキャンして得られた 画像から蛍光強度を算出し、ロゼッタ社製の解析ソフトウェア (Luminator Ver. 1.0)に て解析し、反復実験全てを統計的信頼度 99.999%にて統合し、 5年生のカマルドレン シス種の榭幹基部と榭幹中央部、グロプルス種の榭幹基部における選抜した 59クロ ーンの発現情報を得た。  [0164] In accordance with the RNA extraction method shown in Example 1, the present inventors extracted total RNA from the trunk base and middle part of a 5-year-old Camaldrensis species, and Groplus species. At this time, the crust side (phloem) and the wood fiber-forming tissue side (xylem) of each plant were used. The total RNA power obtained from each individual was purified by the PolyATract mRNA Isolation System manufactured by Promega. Each cRNA obtained by synthesizing a total of 4 types of mRNA obtained in this way was labeled with 2 types of fluorescent dyes (cy3.cy5), and used as probes in oligo microarray analysis. An analysis was performed (Figures 1-3). The method of hybridization including the label followed the analysis protocol presented by Agilent Technologies. Fluorescence intensity is calculated from the images obtained by scanning and analyzed using Rosetta's analysis software (Luminator Ver. 1.0), and all repeated experiments are integrated with a statistical reliability of 99.999%. We obtained information on the expression of 59 clones selected at the trunk base of the cis species and at the center of the trunk, and at the trunk base of the groprus species.
[0165] ユーカリの木繊維細胞壁の形成に関わる遺伝子の発現は、榭皮側（師部）と比較し て木繊維形成組織側 (木部)で有意に発現が強くなると予想される。選抜した 59クロ ーンの中で、有意に木繊維形成組織側で強!、発現を示すクローンを選抜した結果、[0165] Expression of genes involved in the formation of eucalyptus tree fiber cell walls is expected to be significantly stronger on the wood fiber-forming tissue side (xylem) than on the crust side (phloem). Selected 59 black Among the trees, significantly stronger on the side of the wood fiber forming tissue !, as a result of selecting clones showing expression,
34クローンをユーカリの木繊維細胞壁形成に関与している遺伝子群として同定した ( 表 1〜4)。 34 clones were identified as genes involved in Eucalyptus tree fiber cell wall formation (Tables 1-4).
[表 1] [table 1]
Figure imgf000073_0001
Figure imgf000073_0001
上記 Sequence IDは上から順に、配列番号： 1〜34に対応する。 The Sequence ID corresponds to SEQ ID NOs: 1-34 in order from the top.
CesA:Cellulose synthase, SuSy: Sucrose Synthase, EGase: endo— 1,4— beta— glucanase ， DL : Dynamin-like protein, CAD: cinnamyl alcohol dehydrogenase, OMT:〇— methyl transferase, 4CL: 4— coumarate Co A ligase, CCR: Cinnamoyl Co A reductase, C4H: cirmamate 4— hydroxylase CesA: Cellulose synthase, SuSy: Sucrose Synthase, EGase: endo— 1,4— beta— glucanase, DL: Dynamin-like protein, CAD: cinnamyl alcohol dehydrogenase, OMT: 〇— methyl transferase, 4CL: 4— coumarate Co A ligase , CCR: Cinnamoyl Co A reductase, C4H: cirmamate 4— hydroxylase
マイクロアレイ実験 5y-cam5 X vs Pは、 5年生ユーカリ ·カマルドレンシス樹幹中央 部の木部と師部を比較した実験を、 5y-caml X vs Pは 5年生ユーカリ ·カマルドレンシ ス基部の木部と師部を比較した実験を、 5y-glo X vs Pは、 5年生ユーカリ 'グロブルス 基部の木部と師部を比較した実験を表す。実験結果は、木繊維形成組織側と榭皮 側を比較して、前者で強い場合は正の倍数で、榭皮側で強い場合は負の倍数で表 している。 The microarray experiment 5y-cam5 X vs P is an experiment comparing the xylem and phloem of the middle part of the fifth-year eucalyptus / camaldolensis trunk, and 5y-caml X vs P Experiment comparing phloem, 5y-glo X vs P, 5th grade Eucalyptus' Grobulus An experiment comparing the xylem and phloem of the base. The experimental results are expressed as a positive multiple when the former is strong, and as a negative multiple when strong on the husk side, comparing the wood fiber-forming tissue side and the husk side.
[0168] 34個の遺伝子群は、 5年生のカマルドレンシス種の榭幹基部と中央部における詳 細な発現様式から、以下の（1)〜（3)に大別された (表 2、 4)。  [0168] The 34 gene groups were broadly classified into the following (1) to (3) based on the detailed expression patterns in the trunk base and the central part of the fifth-grade Camaldrensis species (Table 2, Four).
[0169] [表 2] [0169] [Table 2]
Figure imgf000074_0001
Figure imgf000074_0001
(1) 26個の「ユーカリ属カマルドレンシスの榭幹基部および榭幹中央部において、師 部側と比較して木部側で発現レベルが上昇している遺伝子群」（配列番号： 1〜6、 8 、 9、 11〜14、 16、 17、 19〜25、 27、 28、 30、 32、 33) (1) 26 “gene groups whose expression level is increased on the xylem side compared to the phloem side in the trunk base and middle trunk of Eucalyptus genus Camaldrensis” (SEQ ID NO: 1 to 6, 8 (9, 11-14, 16, 17, 17, 19-25, 27, 28, 30, 32, 33)
26個の遺伝子群には、榭幹基部または榭幹中央部のいずれかにおいて、師部側と 比べ木部側での発現差力 ¾倍以上である遺伝子群 (配列番号： 1〜6、 9、 11〜14、 1 6、 17、 19、 20、 22、 25、 27、 30)、樹幹基咅また ίま樹幹中央咅の!ヽずれ力にお!/ヽ て、師部側と比べ木部側での発現差が 4倍以上である遺伝子群 (配列番号： 1、 3〜6 、 9、 11〜14、 16、 17、 19、 22、 25、 30)、樹幹基咅また ίま樹幹中央咅の!ヽずれ力 において、師部側と比べ木部側での発現差力 倍以上である遺伝子群 (配列番号： 1 、 3〜6、 11、 12、 14、 16、 17、 22、 25)、樹幹基咅また ίま樹幹中央咅の!/、ずれ力に おいて、師部側と比べ木部側での発現差が 10倍以上である遺伝子群 (配列番号： 1 ゝ 3、 5、 12、 25)力含まれる。  The 26 gene groups include gene groups (SEQ ID NOs: 1 to 6, 9) that have an expression differential power of ¾ times or more on the xylem side compared to the phloem side at either the trunk base or the trunk trunk center. , 11-14, 1 6, 17, 19, 20, 22, 25, 27, 30), the trunk of the trunk or the center of the trunk of the tree! Gene group (SEQ ID NOs: 1, 3 to 6, 9, 11 to 14, 16, 17, 19, 22, 25, 30), stem base or trunk A group of genes (SEQ ID NOs: 1, 3-6, 11, 12, 14, 16, 17, 22, which are more than double the differential force on the xylem side compared to the phloem side in the! 25), a group of genes (SEQ ID NO: 1 ゝ 3, 5, 12, 25) Power included.
[0170] 26個の遺伝子について、榭幹基部と榭幹中央部の木部側での遺伝子発現を直接 比較した結果、 2個（表 4の垂直変動分類グループ A-1)の「榭幹中央部の木部側と 比較して榭幹基部の木部側で発現が上昇している遺伝子群」（配列番号: 33、 34)、 6個（表 4の垂直変動分類グループ Α-2)の「榭幹基部の木部側と榭幹中央部の木部 側での発現が同等な遺伝子群」（配列番号： 9、 11〜13、 23、 27)、 18個（表 4の垂 直変動分類グループ Α-3)の「榭幹基部の木部側と比較して榭幹中央部の木部側で 発現が上昇している遺伝子群」（配列番号： 1〜6、 8、 14、 16、 17、 19〜22、 24、 2 5、 28、 30)に大別された。より具体的には、実験によって得られた平均蛍光強度の 数値を、榭幹基部 Ζ中央部または中央部 Ζ榭幹基部の式により計算した。有意であ る各遺伝子の発現情報から、榭幹基部 Ζ中央部の式によって得られる数値が 1.1以 上かつ Ρく =0.001であるものを「榭幹中央部の木部側と比較して榭幹基部の木部側で 発現が上昇している遺伝子群」と、中央部 Ζ榭幹基部の式によって得られる数値が 1 .1以上かつ Ρく =0.001であるものを「榭幹基部の木部側と比較して榭幹中央部の木部 側で発現が上昇して 、る遺伝子群」と、有意でな 、もしくは平均強度がほぼ同じもの (中央部の木部と基部の木部の蛍光強度比力 Sl.0〜l.l倍未満または Ρ〉0.001)を「榭 幹基部の木部側と榭幹中央部の木部側での発現が同等な遺伝子群」と判断し、分 した。 [0170] As a result of direct comparison of the gene expression on the xylem side of the trunk base and the central trunk of the 26 genes, two (vertical variation classification group A-1 in Table 4) Gene group whose expression is increased on the xylem side of the trunk base compared to the xylem side of the cervical region ”(SEQ ID NO: 33, 34), 6 (vertical variation classification group Α-2 in Table 4) “A group of genes with the same expression on the xylem side of the trunk base and the xylem side of the trunk trunk” (SEQ ID NOs: 9, 11-13, 23, 27), 18 (vertical variation in Table 4) Classification group Α-3) “Gene group whose expression is increased on the xylem side of the trunk trunk compared to the xylem side of the trunk trunk” (SEQ ID NOs: 1-6, 8, 14, 16 17, 19-22, 24, 25, 28, 30). More specifically, the numerical value of the average fluorescence intensity obtained by the experiment was calculated by the formula of the trunk base or the central part or the central part of the trunk base. Based on the expression information of each gene that is significant, the value obtained by the formula of the trunk base and the central part is 1.1 or higher and the value of 0.001 is compared with the xylem side of the central trunk part. `` Gene group whose expression is increased on the xylem side of the trunk base '' and the value obtained by the formula of the trunk trunk base in the center is 1.1 or more and = = 0.001 The gene group has a significant or almost the same average intensity as that of the gene group whose expression is increased on the xylem side of the trunk trunk compared to the root side (the xylem of the central part and the xylem of the base part) The fluorescence intensity specific force Sl. 0 to less than ll times or Ρ> 0.001) was judged as “a group of genes with the same expression on the xylem side of the trunk base and the xylem side of the trunk trunk”.
[0171] 2個の遺伝子群には、榭幹基部の木部と榭幹中央部の木部での発現差が 1.5倍以 上である遺伝子群 (配列番号： 33)が含まれる。 [0171] In the two gene groups, the difference in expression between the xylem of the trunk trunk and the xylem of the trunk trunk is 1.5 times or more. The above gene group (SEQ ID NO: 33) is included.
18個の遺伝子群には、榭幹基部の木部と榭幹中央部の木部での発現差が 1.5倍以 上である遺伝子群（配列番号： 1〜6、 14、 16、 17、 19〜22、 25、 28、 30)、榭幹基 部の木部と榭幹中央部の木部での発現差力 ^倍以上である遺伝子群 (配列番号：1 〜6、 14、 16、 17、 22、 25、 30)、榭幹基部の木部と榭幹中央部の木部での発現差 力^倍以上である遺伝子群 (配列番号： 3、 5、 25、 30)が含まれる。  The 18 gene groups include gene groups (SEQ ID NOs: 1 to 6, 14, 16, 17, 19) that have a difference in expression between the xylem of the trunk trunk and the xylem of the trunk trunk of 1.5 times or more. -22, 25, 28, 30), gene group (SEQ ID NO: 1 to 6, 14, 16, 17, 22, 25, 30), and a group of genes (SEQ ID NOs: 3, 5, 25, 30) that have an expression differential power of at least twice that between the xylem of the trunk base and the xylem of the central trunk.
[0172] (2) 1個（表 4の垂直変動分類グループ D)の「ユーカリ属カマルドレンシスの榭幹基部 において、師部側と比較して木部側で発現レベルが減少し、榭幹中央部において、 師部側と比較して木部側で発現レベルが上昇している遺伝子群」（配列番号： 15) [0172] (2) In the trunk base of Eucalyptus genus Camaldrensis, the expression level decreased on the xylem side compared to the phloem side, and one trunk (vertical variation classification group D in Table 4) In the central part, the gene group whose expression level is increased on the xylem side compared to the phloem side "(SEQ ID NO: 15)
[0173] (3) 6個（表 4の垂直変動分類グループ G)の「ユーカリ属カマルドレンシスの榭幹中央 部において、師部側と比較して木部側で発現レベルが上昇し、榭幹基部において、 師部側と木部側で発現レベルが同等な遺伝子群」（配列番号： 7、 10、 18、 26、 29、 34) [0173] (3) The expression level increased in the xylem side in the middle of the trunk of Eucalyptus genus Camaldrensis compared to the phloem side in 6 (vertical variation classification group G in Table 4). In the trunk base, a group of genes whose expression levels are the same on the phloem side and the xylem side ”
6個の遺伝子群には、榭幹中央部において、師部側と比べて木部側での発現差が 2倍以上である遺伝子群 (配列番号： 7、 10、 18、 26、 29)、榭幹中央部において、 師部側と比べて木部側での発現差が 2.5倍以上である遺伝子群 (配列番号： 18、 26 、 29)が含まれる。  The six gene groups have gene groups (SEQ ID NOs: 7, 10, 18, 26, 29) whose expression difference on the xylem side is more than double in the middle of the trunk, compared to the phloem side, In the center of the trunk, the gene group (SEQ ID NO: 18, 26, 29) whose expression difference on the xylem side is 2.5 times or more compared to the phloem side is included.
34個の遺伝子群は、 5年生のカマルドレンシス種の榭幹基部または榭幹中央部と 5 年生のグロプルス種の榭幹基部における詳細な発現様式から、以下の（1)〜（3)に 大別された (表 3、 4)。  The 34 gene groups are expressed in the following (1) to (3) from the detailed expression pattern in the trunk base of the fifth-grade Camaldorensis species or the middle of the trunk and the fifth-grade Groplus species. Broadly classified (Tables 3 and 4).
[0174] [表 3] [0174] [Table 3]
Figure imgf000077_0001
4]
Figure imgf000077_0001
Four]
Sequence 分類グループ 分類グループ ID 一次配列名 注釈 Sequence classification group Classification group ID Primary sequence name Comments
(垂直変動） (樹種間変動） (Vertical fluctuation) (Variation between trees)
72242 Stem_001J00035B07 CesA A - 3 E72242 Stem_001J00035B07 CesA A-3 E
74993 Stem— 001 J05J. CL.16. cl.1645. Conti l CesA A - 3 E74993 Stem— 001 J05J. CL.16. Cl.1645. Conti l CesA A-3 E
73371 Sten_001_005_1. CL.1. cl.183. Contig2 CesA A - 3 E73371 Sten_001_005_1. CL.1. Cl.183. Contig2 CesA A-3 E
70577 Stem_001J05_1. CL.7. cl.746. Contig2 CesA A - 3 E70577 Stem_001J05_1. CL.7. Cl.746. Contig2 CesA A-3 E
74689 Stem— 001— 005—1. CL.1. cl.183. Contigl CesA A - 3 E74689 Stem— 001— 005—1. CL.1. Cl.183. Contigl CesA A-3 E
77018 Stem— 001— 005— 1. CL.0. cl.065. Cont igl CesA A - 3 E77018 Stem— 001— 005— 1. CL.0. Cl.065. Cont igl CesA A-3 E
71104 Stem_001_000236C09 CesA G I71104 Stem_001_000236C09 CesA G I
74384 Stem— 001— 005— 1. CL.0. cl.005. Conti 4 SuSy A - 3 C74384 Stem— 001— 005— 1. CL.0. Cl.005. Conti 4 SuSy A-3 C
73919 Stem_001_005_1. CL.26. cl.2656. Contigl SuSy A-2 E73919 Stem_001_005_1. CL.26. Cl.2656. Contigl SuSy A-2 E
73060 SteiOOl— 005— 1. CL.5. cl.554. Contigl EGase G I73060 SteiOOl— 005— 1. CL.5. Cl.554. Contigl EGase G I
72025 StemJ01_005_1. CL.1. cl.188. Contigl Chi tinase A-2 A-172025 StemJ01_005_1. CL.1. Cl.188. Contigl Chi tinase A-2 A-1
76261 Stem_005_000020H03 Chitinase A-2 A-176261 Stem_005_000020H03 Chitinase A-2 A-1
74559 Stera—001— 005— 1. CL 24. cl.2499. Cont igl DL A-2 C74559 Stera—001— 005— 1. CL 24. cl.2499. Cont igl DL A-2 C
76163 Stem— 001— 000199B12 CAD A - 3 E76163 Stem— 001— 000199B12 CAD A-3 E
76841 SteiOOl— 005—1. CL.17. cl.1796. Contigl CAD D 176841 SteiOOl— 005—1. CL.17. Cl.1796. Contigl CAD D 1
72145 Stem— 001— 005—1. CL.6. cl.668. Contigl CAD A - 3 E72145 Stem— 001— 005—1. CL.6. Cl.668. Contigl CAD A-3 E
75604 Stem— 001— 005— 1. CL.0. cl.054. ContigZ 0 T A - 3 A-175604 Stem— 001— 005— 1. CL.0. Cl.054. ContigZ 0 T A-3 A-1
74169 Stem— 001— 005—1. CL.1. cl.103. Contigl OMT G 174169 Stem— 001— 005—1. CL.1. Cl.103. Contigl OMT G 1
74309 Stem_001_005_1. CL.1. cl.103. Contig3 OWT A-3 C74309 Stem_001_005_1.CL.1.cl.103.Contig3 OWT A-3 C
73368 Stem_001_000002H07 OMT A-3 E73368 Stem_001_000002H07 OMT A-3 E
70874 Stem_001_000013E05 OMT A-3 A-370874 Stem_001_000013E05 OMT A-3 A-3
73879 Stem— 001— 005— 1. CL.0. cl.078. Contigl 4CL A-3 A-173879 Stem— 001— 005— 1. CL.0. Cl.078. Contigl 4CL A-3 A-1
70484 Stem_001_005_1. CL.21. cl.2195. Contigl CCR A-2 C70484 Stem_001_005_1.CL.21.cl.2195.Contigl CCR A-2 C
73946 Stem_001_000184C07 C4H A-3 C73946 Stem_001_000184C07 C4H A-3 C
72892 Stem—001一 005— 1. CL.7. cl.735. Contigl Laccase A-3 C72892 Stem—001 1 005— 1. CL.7. Cl.735. Contigl Laccase A-3 C
75489 Stem— 001—005—1. CL.9. cl.967. Contigl Laccase G I75489 Stem— 001—005—1. CL.9. Cl.967. Contigl Laccase G I
77263 Stem— 001 JJ05—1. CL.9. cl.949. Contigl Laccase A-2 E77263 Stem— 001 JJ05—1. CL.9. Cl.949. Contigl Laccase A-2 E
74948 Stem— 001—005— 1. CL.10. cl.1024. Contigl Laccase A-3 E74948 Stem— 001—005— 1. CL.10. Cl.1024. Contigl Laccase A-3 E
76820 Stem— 001— 000132G06 Laccase G I76820 Stem— 001— 000132G06 Laccase G I
77892 Stem_005_000016E07 Laccase A-3 A-477892 Stem_005_000016E07 Laccase A-3 A-4
74825 Stem_005_000016C03 Peroxidase 1 174825 Stem_005_000016C03 Peroxidase 1 1
75691 Stem_001_005_1. CL 2. cl.284. Contigl Peroxidase A-1 E75691 Stem_001_005_1. CL 2. cl.284. Contigl Peroxidase A-1 E
72698 Stem— 005— 000018H09 Peroxidase A-1 C72698 Stem— 005— 000018H09 Peroxidase A-1 C
74221 Stem—001— 005—1. CL.11. cl.1102. Contigl Peroxidase G 1 74221 Stem—001— 005—1. CL.11. Cl.1102. Contigl Peroxidase G 1
(1) 6個の「ユーカリ属カマルドレンシスおよびユーカリ属グロブルスの榭幹において、 師部側と比較して木部側で発現レベルが上昇している遺伝子群」（配列番号： 11、 1 2、 17、 21、 22、 30) (1) Six “groups of genes whose expression level is increased on the xylem side compared to the phloem side in the trunk of Eucalyptus camaldrensis and Eucalyptus globulus” (SEQ ID NOs: 11, 1 2 , 17, 21, 22, 30)
6個の遺伝子群には、ユーカリ属カマルドレンシスおよびユーカリ属グロブルスの榭 幹の 、ずれかにお 、て、師部側と比べ木部側での発現差が 4倍以上である遺伝子群 (配列番号： 11、 12、 17、 22、 30)、ユーカリ属カマルドレンシスおよびユーカリ属グ ロブルスの榭幹のいずれかにおいて、発現差が 6倍以上である遺伝子群 (配列番号： 11、 12、 17、 22)、ユーカリ属カマルドレンシスおよびユーカリ属グロブルスの榭幹 のいずれかにおいて、発現差が 8倍以上である遺伝子群 (配列番号： 11、 12、 17)、 ユーカリ属カマルドレンシスおよびユーカリ属グロブルスの榭幹の 、ずれかにお！/、て 、発現差が 10倍以上である遺伝子群 (配列番号：12)が含まれる。 The six gene groups include genes that have a difference in expression of eucalyptus chamaldrensis and eucalyptus globulus stems that are more than 4 times different in the xylem compared to the phloem. SEQ ID NOs: 11, 12, 17, 22, 30), Eucalyptus Camaldrensis and Eucalyptus In any of the trunks of Robles, expression in any of the trunks of genes (SEQ ID NOs: 11, 12, 17, 22), Eucalyptus genus Camaldrensis and Eucalyptus globus The gene group (SEQ ID NOs: 11, 12, 17) with a difference of more than 8 times, Eucalyptus genus Camaldrensis and Eucalyptus globules trunks! Thus, a gene group (SEQ ID NO: 12) whose expression difference is 10 times or more is included.
[0176] 6個の遺伝子について、ユーカリ属カマルドレンシスの榭幹基部または榭幹中央部 の木部側とユーカリ属グロブルスの榭幹基部の木部側での遺伝子発現を直接比較し た結果、 4個（表 4の榭種間変動分類グループ A-1)の「ユーカリ属グロブルスの榭幹 の木部側と比較してユーカリ属カマルドレンシスの樹幹の木部側で発現が上昇して いる遺伝子群」（配列番号： 11、 12、 17、 22)、 1個（表 4の榭種間変動分類グループ A-3)の「ユーカリ属グロブルスの榭幹の木部側と比較してユーカリ属カマルドレンシ スの榭幹の木部側で発現が減少している遺伝子群」（配列番号 : 21)に大別された。 より具体的には、実験によって得られた平均蛍光強度の数値を、ユーカリ属カマルド レンシスの榭幹 (基部または中央部）の木部 Zユーカリ属グロブルスの榭幹 (基部）の 木部の式またはユーカリ属グロブルスの榭幹 (基部）の木部 Zユーカリ属カマルドレ ンシスの榭幹 (基部または中央部）の木部の式により計算した。各遺伝子の発現情報 力 、ユーカリ属カマルドレンシス Zユーカリ属グロブルスの榭幹の木部の式によって 得られる数値が 1.1以上かつ P〈=0.001であるものを「ユーカリ属グロブルスの樹幹の 木部側と比較してユーカリ属カマルドレンシスの樹幹の木部側で発現が上昇している 遺伝子群」と、ユーカリ属グロブルスの榭幹 Zユーカリ属カマルドレンシスの木部の式 によって得られる数値が 1.1以上かつ P〈=0.001であるものを「ユーカリ属カマルドレン シスの榭幹の木部側と比較してユーカリ属グロブルスの樹幹の木部側で発現が上昇 して 、る遺伝子群」と、有意でな 、もしくは平均強度がほぼ同じもの（ユーカリ属グロ ブルスの榭幹の木部に比べてユーカリ属カマルドレンシスの樹幹の木部の蛍光強度 比力 l.0〜l.l倍未満または P〉0.001)を「ユーカリ属カマルドレンシスの樹幹の木部側 とユーカリ属グロブルスの樹幹の木部側での発現が同等な遺伝子群」と判断し、分類 した。 [0176] As a result of direct comparison of the gene expression on the xylem side of the trunk base of Eucalyptus genus Camaldrensis or the central part of eucalyptus globules and the xylem side of the trunk base of Eucalyptus globulus for six genes, The expression is increased on the xylem side of the Eucalyptus chamaldrensis trunk compared to the xylem side of the Eucalyptus globulus trunk in four (Table 4 varietal variation classification group A-1 in Table 4) Gene group ”(SEQ ID NO: 11, 12, 17, 22), 1 (variety group A-3 in Table 4 varieties classification group A-3)“ Eucalyptus genus compared to the xylem side of Eucalyptus globulus trunk It was broadly classified into “gene groups whose expression is reduced on the xylem side of the trunk of camaldrensis” (SEQ ID NO: 21). More specifically, the numerical value of the average fluorescence intensity obtained by the experiment is calculated based on the xylem formula of the trunk (base or central part) of the Eucalyptus genus Camaldrensis Z Eucalyptus globulus trunk (base) xylem Z Eucalyptus chamaldorensis trunk (base or middle) xylem calculation. Expression information power of each gene, Eucalyptus genus Camaldrensis Z Eucalyptus globules trunk xylem value is 1.1 or more and P <= 0.001 The number of genes obtained by the expression of the xylem of the eucalyptus chamaldrensis tree and the eucalyptus globulus trunk Z As described above, P <= 0.001 is significantly different from `` the gene group in which the expression is increased on the xylem side of the Eucalyptus globulis tree compared to the xylem side of the Eucalyptus camaldrensis ''. Or the average intensity is almost the same (the fluorescence intensity of the tree part of Eucalyptus genus Camaldrensis relative to the tree part of Eucalyptus globulus trunk is less than l.0 to ll times or P> 0.001) `` Yu -It was classified as a gene group with the same expression on the xylem side of the trunk of the genus Cali genus Camaldrensis and on the xylem side of the stem of the Eucalyptus globules.
[0177] 4個の遺伝子群には、ユーカリ属カマルドレンシスの木部とユーカリ属グロブルスの 木部での発現差力 倍以上である遺伝子群 (配列番号： 12、 17)、ユーカリ属カマル ドレンシスの木部とユーカリ属グロブルスの木部での発現差が 9倍以上である遺伝子 群 (配列番号： 12)が含まれる。 [0177] There are four gene groups in the xylem of the Eucalyptus Camaldrensis and the Eucalyptus Globulus. Gene group (SEQ ID NOs: 12, 17) that is more than double the differential expression in xylem, Gene group (sequence) that is more than 9-fold differential in xylem of Eucalyptus chamaldrensis and Eucalyptus globulus Number: 12) is included.
[0178] (2) 7個（表 4の榭種間変動分類グループ C)の「ユーカリ属カマルドレンシスの樹幹に おいて、師部側と比較して木部側で発現レベルが上昇し、ユーカリ属グロブルスの榭 幹において、師部側と比較して木部側で発現レベルが減少している遺伝子群」（配 列番号： 8、 13、 19、 23〜25、 33)  [0178] (2) The expression level on the xylem side increased compared to that on the phloem side in the trunk of Eucalyptus genus Camaldrensis, which was 7 (Table 4 varietal variation classification group C in Table 4) In the trunk of the Eucalyptus globules, the gene group whose expression level is reduced on the xylem side compared to the phloem side "(sequence numbers: 8, 13, 19, 23-25, 33)
7個の遺伝子群には、ユーカリ属カマルドレンシスの榭幹において、師部側と比べ 木部側での発現差が 2.5倍以上、またはユーカリ属グロブルスの榭幹において、木部 側と比べ師部側での発現差が 2.5倍以上である遺伝子群 (配列番号: 8、 13、 19、 24 、 25、 33)、ユーカリ属カマルドレンシスの榭幹において、師部側と比べ木部側での 発現差が 4倍以上、またはユーカリ属グロブルスの榭幹において、木部側と比べ師部 側での発現差が 4倍以上である遺伝子群 (配列番号： 13、 19、 25)ユーカリ属カマル ドレンシスの榭幹において、師部側と比べ木部側での発現差が 10倍以上、またはュ 一力リ属グロブルスの樹幹にぉ 、て、木部側と比べ師部側での発現差が 10倍以上で ある遺伝子群 (配列番号： 25)が含まれる。  The seven gene groups show that the difference in the expression on the xylem side is 2.5 times or more in the trunk of Eucalyptus genus Camaldrensis, or in the trunk of Eucalyptus globulus compared with the xylem side. Gene groups (SEQ ID NOs: 8, 13, 19, 24, 25, 33) that have an expression difference of 2.5 times or more on the cervical side, in the trunk of the Eucalyptus genus Camaldrensis, on the xylem side compared to the phloem side Gene group (SEQ ID NOs: 13, 19, 25) Eucalyptus Kamal whose expression difference is 4 times or more, or in Eucalyptus globulus trunks, the expression difference on the phloem side is 4 times or more compared to the xylem side In the trunk of Drensis, the expression difference on the xylem side is more than 10 times compared to the phloem side, or the expression difference on the phloem side compared to the xylem side, A gene group (SEQ ID NO: 25) that is 10 times or more is included.
[0179] (3) 13個（表 4の榭種間変動分類グループ E)の「ユーカリ属カマルドレンシスの榭幹 において、師部側と比較して木部側で発現レベルが上昇し、ユーカリ属グロブルスの 榭幹において、師部側と木部側で発現レベルが同等な遺伝子群」（配列番号：1〜6 、 9、 14、 16、 20、 27、 28、 32)  [0179] (3) In the trunk of Eucalyptus genus Camaldrensis, the expression level increased on the xylem side compared to the phloem side, and 13 eucalyptus In the trunk of the genus Globulus, genes with similar expression levels on the phloem side and the xylem side ”(SEQ ID NOs: 1-6, 9, 14, 16, 20, 27, 28, 32)
13個の遺伝子群には、ユーカリ属カマルドレンシスの榭幹において、師部側と比べ て木部側での発現差が 3倍以上である遺伝子群 (配列番号： 1〜6、 9、 14、 16、 20、 27)、ユーカリ属カマルドレンシスの榭幹において、師部側と比べて木部側での発現 差が 2倍以上である遺伝子群 (配列番号： 1、 3〜6、 14、 16)、ユーカリ属カマルドレ ンシスの榭幹にお 、て、師部側と比べて木部側での発現差が 10倍以上である遺伝 子群 (配列番号： 1、 3、 5)が含まれる。  The 13 gene groups include gene groups (SEQ ID NOs: 1-6, 9, 14) that have a three-fold or more differential expression on the xylem side compared to the phloem side in the trunk of the Eucalyptus genus Camaldrensis. , 16, 20, 27) In Eucalyptus chamaldrensis trunks, the gene group is more than double the expression difference on the xylem side compared to the phloem side (SEQ ID NO: 1, 3-6, 14) 16), Eucalyptus genus Camaldrensis trunks include a group of genes (SEQ ID NOs: 1, 3, 5) whose expression difference on the xylem side is more than 10 times that of the phloem side It is.
[0180] (実施例 4)木繊維細胞壁形成に関わる遺伝子群のゲノムクローンとプロモーター DN Aの取得 (1)ユーカリ BACゲノムライブラリーのスクリーニング [0180] (Example 4) Acquisition of genomic clones and promoter DNA of genes involved in wood fiber cell wall formation (1) Eucalyptus BAC genomic library screening
ユーカリ BACゲノムライブラリー作製は、 D. Petersonらの J. Agricultural Genomics 5 (http：〃 www.ncgr.orc/research/jag)の方法に従った。作製したライブラリ一は、個 別クローン毎に 384ゥエルプレート 120枚に分別した。これらのプレートから、プレート 毎、全プレートの各列、各行の DNAを調製し、 PCRによる 3Dスクリーニング用の DNA プールとした。 cDNA配列情報を基に合成した配列番号： 69〜84のオリゴヌクレオチ ドプライマ一（配列番号： 69〜84の奇数番号はフォワードプライマー、偶数番号はリ バースプライマー）を用い、 DNAプールを铸型に PCR法により各遺伝子のゲノムクロ ーンのスクリーニングを行った。  Eucalyptus BAC genomic library was prepared according to the method of J. Agricultural Genomics 5 (http: 〃 www.ncgr.orc / research / jag) by D. Peterson et al. The prepared library was sorted into 120 384 well plates for each individual clone. From these plates, DNA was prepared for each plate, each column and each row of all plates, and used as a DNA pool for 3D screening by PCR. PCR using an oligonucleotide primer of SEQ ID NO: 69-84 synthesized based on cDNA sequence information (odd number of SEQ ID NO: 69-84 is a forward primer and even number is a reverse primer). The genomic clones of each gene were screened by the method.
[0181] (2)ゲノム配列の決定 [0181] (2) Determination of genome sequence
BACクローンのインサート配列は、実施例 1に示す方法で決定した。得られた塩基 配列を用い、 cDNA配列をもとに各遺伝子のゲノム領域を決定した (配列番号： 3、 6、 11、 14、 19、 23、 24、 25に記載の cDNA酉己列をもとに決定されたゲノム酉己列を、そ れぞれ、配列番号： 85〜92に示す)。また、翻訳開始コドン ATG (アミノ酸ではメチォ ニンをコード）を含む全長の cDNA配列であったクローンに関しては、その配列をもと にプロモーター領域を決定した。決定されたプロモーター配列を配列番号： 93〜 10 0に示した。具体的には、全長 cDNAの 5 '末端塩基から上流の配列を、遺伝情報処 理ソフトウェア「GENETYX」（ソフトウェア開発株式会社）により解析し、 TATA-box配 列を決定した。高等真核生物のプロモーター領域には、転写開始点より通常約 10〜 25塩基上流側に TATA-box配列があることがよく知られて!/、る。決定された TATA-bo Xの位置カゝら全長 cDNA5 '末端塩基までが近接している（約 lOObp程度）場合、 cDN Aはほぼ完全長であり、その末端塩基上流側がプロモーター領域であると判断した。 プロモーターの長さは、モデル植物で知られている最大の長さ（約 3kb)を参考に、 c DNA5 '末端塩基から上流側最大 3kbまでとした。  The insert sequence of the BAC clone was determined by the method shown in Example 1. Using the obtained base sequence, the genomic region of each gene was determined based on the cDNA sequence (the cDNA self-sequences described in SEQ ID NOs: 3, 6, 11, 14, 19, 23, 24, 25 were also used). And the determined genome sequences are shown in SEQ ID NOs: 85 to 92, respectively). For clones that were full-length cDNA sequences including the translation initiation codon ATG (which encodes methionine in amino acids), the promoter region was determined based on that sequence. The determined promoter sequence is shown in SEQ ID NOs: 93-100. Specifically, the TATA-box sequence was determined by analyzing the sequence upstream from the 5 ′ end base of the full-length cDNA using the genetic information processing software “GENETYX” (Software Development Co., Ltd.). It is well known that the promoter region of higher eukaryotes has a TATA-box sequence usually about 10 to 25 bases upstream from the transcription start site! If the position of the determined TATA-bo X is close to the full-length cDNA 5 'terminal base (approx. LOObp), it is judged that cDNA is almost full length and that the upstream side of the terminal base is the promoter region. did. The length of the promoter was set to a maximum of 3 kb upstream from the 5′-terminal base of cDNA with reference to the maximum length (about 3 kb) known in model plants.
[0182] 配列番号： 1によって示される塩基配列では、第 1位〜第 660位がアミノ酸コード領 域 (配列番号： 35)である。 [0182] In the nucleotide sequence represented by SEQ ID NO: 1, positions 1 to 660 are the amino acid coding region (SEQ ID NO: 35).
配列番号： 2によって示される塩基配列では、第 1位〜第 602位がアミノ酸コード領 域 (配列番号： 36)である。 配列番号： 3によって示される塩基配列では、第 337位〜第 3273位がアミノ酸コー ド領域 (配列番号: 37)である。 In the base sequence represented by SEQ ID NO: 2, the 1st to 602nd positions are the amino acid coding region (SEQ ID NO: 36). In the base sequence represented by SEQ ID NO: 3, the 337th to 3273th positions are the amino acid code region (SEQ ID NO: 37).
配列番号: 4によって示される塩基配列では、第 1位〜第 1407位がアミノ酸コード 領域 (配列番号: 38)である。  In the base sequence represented by SEQ ID NO: 4, positions 1 to 1407 are the amino acid coding region (SEQ ID NO: 38).
配列番号： 5によって示される塩基配列では、第 1位〜第 465位がアミノ酸コード領 域 (配列番号： 39)である。  In the base sequence represented by SEQ ID NO: 5, the 1st to 465th positions are the amino acid coding region (SEQ ID NO: 39).
配列番号: 6によって示される塩基配列では、第 87位〜第 3221位がアミノ酸コード 領域 (配列番号: 40)である。  In the base sequence represented by SEQ ID NO: 6, the 87th to 3221th positions are the amino acid coding region (SEQ ID NO: 40).
配列番号： 7によって示される塩基配列では、第 1位〜第 699位がアミノ酸コード領 域 (配列番号: 41)である。  In the base sequence represented by SEQ ID NO: 7, the 1st to 699th positions are the amino acid coding region (SEQ ID NO: 41).
配列番号: 8によって示される塩基配列では、第 1位〜第 1407位がアミノ酸コード 領域 (配列番号: 42)である。  In the base sequence represented by SEQ ID NO: 8, positions 1 to 1407 are the amino acid coding region (SEQ ID NO: 42).
配列番号： 9によって示される塩基配列では、第 1位〜第 1122位がアミノ酸コード 領域 (配列番号: 43)である。  In the base sequence represented by SEQ ID NO: 9, positions 1 to 1122 are the amino acid coding region (SEQ ID NO: 43).
配列番号 10によって示される塩基配列では、第 1位〜第 1005位がアミノ酸コード 領 (配列番号: 44)域である。  In the base sequence shown by SEQ ID NO: 10, the 1st to 1005th positions are the amino acid coding region (SEQ ID NO: 44).
配列番号： 11によって示される塩基配列では、第 313位〜第 1305位がアミノ酸コ ード領域 (配列番号： 45)である。  In the base sequence represented by SEQ ID NO: 11, positions 313 to 1305 are the amino acid code region (SEQ ID NO: 45).
配列番号： 12によって示される塩基配列では、第 1位〜第 465位がアミノ酸コード 領域 (配列番号: 46)である。  In the nucleotide sequence represented by SEQ ID NO: 12, positions 1 to 465 are the amino acid coding region (SEQ ID NO: 46).
配列番号： 13によって示される塩基配列では、第 1位〜第 762位がアミノ酸コード 領域 (配列番号: 47)である。  In the base sequence represented by SEQ ID NO: 13, positions 1 to 762 are the amino acid coding region (SEQ ID NO: 47).
配列番号： 14によって示される塩基配列では、第 375位〜第 1442位がアミノ酸コ ード領域 (配列番号： 48)である。  In the nucleotide sequence represented by SEQ ID NO: 14, positions 375 to 1442 are the amino acid code region (SEQ ID NO: 48).
配列番号： 15によって示される塩基配列では、第 1位〜第 774位がアミノ酸コード 領域 (配列番号: 49)である。  In the nucleotide sequence represented by SEQ ID NO: 15, positions 1 to 774 are the amino acid coding region (SEQ ID NO: 49).
配列番号： 16によって示される塩基配列では、第 1位〜第 498位がアミノ酸コード 領域 (配列番号： 50)である。 配列番号： 17によって示される塩基配列では、第 68位〜第 1165位がアミノ酸コー ド領域 (配列番号: 51)である。 In the base sequence represented by SEQ ID NO: 16, positions 1 to 498 are the amino acid coding region (SEQ ID NO: 50). In the base sequence represented by SEQ ID NO: 17, positions 68 to 1165 are the amino acid code region (SEQ ID NO: 51).
配列番号： 18によって示される塩基配列では、第 117位〜第 857位がアミノ酸コー ド領域 (配列番号: 52)である。  In the base sequence represented by SEQ ID NO: 18, the 117th to 857th positions are the amino acid code region (SEQ ID NO: 52).
配列番号： 19によって示される塩基配列では、第 84位〜第 968位がアミノ酸コード 領域 (配列番号: 53)である。  In the nucleotide sequence represented by SEQ ID NO: 19, positions 84 to 968 are the amino acid coding region (SEQ ID NO: 53).
配列番号： 20によって示される塩基配列では、第 1位〜第 207位がアミノ酸コード 領域 (配列番号： 54)である。  In the base sequence represented by SEQ ID NO: 20, positions 1 to 207 are the amino acid coding region (SEQ ID NO: 54).
配列番号： 21によって示される塩基配列では、第 56位〜第 223位がアミノ酸コード 領域 (配列番号： 55)である。  In the base sequence represented by SEQ ID NO: 21, positions 56 to 223 are the amino acid coding region (SEQ ID NO: 55).
配列番号： 22によって示される塩基配列では、第 1位〜第 621位がアミノ酸コード 領域 (配列番号： 56)である。  In the base sequence represented by SEQ ID NO: 22, positions 1 to 621 are the amino acid coding region (SEQ ID NO: 56).
配列番号： 23によって示される塩基配列では、第 323位〜第 1330位がアミノ酸コ ード領域 (配列番号： 57)である。  In the base sequence represented by SEQ ID NO: 23, the 323rd to 1330th positions are the amino acid code region (SEQ ID NO: 57).
配列番号： 24によって示される塩基配列では、第 240位〜第 1847位がアミノ酸コ ード領域 (配列番号： 58)である。  In the nucleotide sequence represented by SEQ ID NO: 24, positions 240 to 1847 are the amino acid code region (SEQ ID NO: 58).
配列番号： 25によって示される塩基配列では、第 273位〜第 1928位がアミノ酸コ ード領域 (配列番号： 59)である。  In the nucleotide sequence represented by SEQ ID NO: 25, the 273rd to 1928th positions are the amino acid code region (SEQ ID NO: 59).
配列番号： 26によって示される塩基配列では、第 1位〜第 1134位がアミノ酸コード 領域 (配列番号: 60)である。  In the base sequence represented by SEQ ID NO: 26, the 1st to 1134th positions are the amino acid coding region (SEQ ID NO: 60).
配列番号： 27によって示される塩基配列では、第 1位〜第 525位がアミノ酸コード 領域 (配列番号: 61)である。  In the base sequence represented by SEQ ID NO: 27, the 1st to 525th positions are the amino acid coding region (SEQ ID NO: 61).
配列番号： 28によって示される塩基配列では、第 1位〜第 1116位がアミノ酸コード 領域 (配列番号: 62)である。  In the base sequence represented by SEQ ID NO: 28, the 1st to 1116th positions are the amino acid coding region (SEQ ID NO: 62).
配列番号： 29によって示される塩基配列では、第 1位〜第 195位がアミノ酸コード 領域 (配列番号: 63)である。  In the base sequence represented by SEQ ID NO: 29, the 1st to 195th positions are the amino acid coding region (SEQ ID NO: 63).
配列番号： 30によって示される塩基配列では、第 1位〜第 138位がアミノ酸コード 領域 (配列番号: 64)である。 配列番号： 31によって示される塩基配列では、第 1位〜第 564位がアミノ酸コード 領域 (配列番号: 65)である。 In the base sequence represented by SEQ ID NO: 30, the first to 138th positions are the amino acid coding region (SEQ ID NO: 64). In the base sequence represented by SEQ ID NO: 31, positions 1 to 564 are the amino acid coding region (SEQ ID NO: 65).
配列番号： 32によって示される塩基配列では、第 1位〜第 711位がアミノ酸コード 領域 (配列番号: 66)である。  In the base sequence represented by SEQ ID NO: 32, the 1st to 711st positions are the amino acid coding region (SEQ ID NO: 66).
配列番号： 33によって示される塩基配列では、第 84位〜第 569位がアミノ酸コード 領域 (配列番号: 67)である。  In the nucleotide sequence represented by SEQ ID NO: 33, the 84th to 569th positions are the amino acid coding region (SEQ ID NO: 67).
配列番号： 34によって示される塩基配列では、第 1位〜第 249位がアミノ酸コード 領域 (配列番号: 68)である。  In the base sequence represented by SEQ ID NO: 34, the 1st to 249th positions are the amino acid coding region (SEQ ID NO: 68).
配列番号： 85によって示される塩基配列の第 1位〜第 2013位がプロモーター領域 (配列番号： 93)であり、第 2350位〜第 2527位、第 3105位〜第 3181位、第 3371 位〜第 3422位、第 3539位〜第 3680位、第 4295位〜第 4561位、第 4924位〜第 5269位、第 5412位〜第 5549位、第 5793位〜第 5918位、第 6001位〜第 6213 位、第 6649位〜第 6907位、第 6993位〜第 7192位、第 7431位〜第 7784位、第 8069位〜第 8650位がコード領域内のエタソンであり、第 2528位〜第 3104位、第 3182位〜第 3370位、第 3423位〜第 3538位、第 3681位〜第 4294位、第 4562 位〜第 4923位、第 5270位〜第 5411位、第 5550位〜第 5792位、第 5919位〜第 6000位、第 6214位〜第 6648位、第 6908位〜第 6992位、第 7193位〜第 7430 位、第 7785位〜第 8068位、がイントロン領域である。  The 1st to 2013th positions of the nucleotide sequence represented by SEQ ID NO: 85 are the promoter region (SEQ ID NO: 93), 2350th to 2527th, 3105th to 3181th, 3371th to 3rd. 3422, 3539-3680, 4295-4561, 4924-5269, 5412-5549, 5793-5918, 6001-6213 6649th to 6907th, 6993th to 7192th, 7431th to 7784th, 8069th to 8650th are Etason in the code region, 2528th to 3104th, 3182-3370, 3423-3538, 3681-4294, 4562-4923, 5270-5411, 5550-5792, 5919 -6th, 6214th to 6648th, 6908th to 6992th, 7193th to 7430th, 7785th to 8068th are intron regions.
配列番号: 86によって示される塩基配列の第 1位〜第 2920位がプロモーター領域 (配列番号： 94)であり、第 3068位〜第 3139位、第 3941位〜第 4141位、第 4275 位〜第 4396位、第 4488位〜第 4554位、第 4670位〜第 4808位、第 5291位〜第 5903位、第 6000位〜第 6137位、第 6426位〜第 6551位、第 6792位〜第 7004 位、第 7091位〜第 7606位、第 7998位〜第 8348位、第 8695位〜第 9276位がコ ード領域内のエタソンであり、第 3140位〜第 3940位、第 4142位〜第 4274位、第 4397位〜第 4487位、第 4555位〜第 4669位、第 4809位〜第 5290位、第 5904 位〜第 5999位、第 6138位〜第 6425位、第 6552位〜第 6791位、第 7005位〜第 7090位、第 7607位〜第 7997位、第 8349位〜第 8694位力イントロン領域である。 配列番号: 87によって示される塩基配列の第 1位〜第 2700位がプロモーター領域 (配列番号： 95)であり、第 3013位〜第 3415位、第 3508位〜第 3667位、第 4610 位〜第 5039位がコード領域内のエタソンであり、第 3416位〜第 3507位、第 3668 位〜第 4609位がイントロン領域である。 The 1st to 2920th positions of the nucleotide sequence represented by SEQ ID NO: 86 are the promoter region (SEQ ID NO: 94), 3068th to 3139th positions, 3941th to 4141th positions, 4275th to 4275th positions. 4396, 4488-4554, 4670-4808, 5291-5903, 6000-6137, 6426-6551, 6792-7004 Nos. 7091 to 7606, 7998 to 8348, 8695 to 9276 are Etasons in the code area, 3140 to 3940, 4142 to 4274. , 4397 to 4487, 4555 to 4669, 4809 to 5290, 5904 to 5999, 6138 to 6425, 6552 to 6791, 7005th to 7090th, 7607th to 7997th, 8349th to 8694th intron region. The 1st to 2700th positions of the nucleotide sequence represented by SEQ ID NO: 87 are the promoter region (SEQ ID NO: 95). Nos. 3013 to 3415, 3508 to 3667, 4610 to 5039 are etasons in the coding region, 3416 to 3507, The 3668th to 4609th positions are intron regions.
配列番号: 88によって示される塩基配列の第 1位〜第 2707位がプロモーター領域 (配列番号： 96)であり、第 3082位〜第 3170位、第 3365位〜第 3478位、第 3565 位〜第 3792位、第 4389位〜第 4828位、第 5124位〜第 5320位がコード領域内 のェクソンであり、第 3171位〜第 3364位、第 3479位〜第 3564位、第 3793位〜 第 4388位、第 4829位〜第 5123位力 Sイントロン領域である。  The 1st to 2707th positions of the base sequence represented by SEQ ID NO: 88 are the promoter region (SEQ ID NO: 96), 3082th to 3170th position, 3365th to 3478th position, 3565th to 3rd position No. 3792, No. 4389 to No. 4828, No. 5124 to No. 5320 are Exons in the code area, No. 3171 to No. 3364, No. 3479 to No. 3564, No. 3793 to No. 4388 4829th to 5123rd position S intron region.
配列番号: 89によって示される塩基配列の第 1位〜第 2835位がプロモーター領域 (配列番号： 97)であり、第 2920位〜第 3011位、第 3142位〜第 3221位、第 3345 位〜第 3489位、第 3643位〜第 3774位、第 4333位〜第 4623位がコード領域内 のェクソンであり、第 3012位〜第 3141位、第 3222位〜第 3344位、第 3490位〜 第 3642位、第 3775位〜第 4332位力 Sイントロン領域である。  The 1st to 2835th positions of the nucleotide sequence represented by SEQ ID NO: 89 are the promoter region (SEQ ID NO: 97), the 2920th to 3011th positions, the 3142th to 3221th positions, the 3345th to 3rd positions. 3489, 3643-3774, 4333-4623 are Exons in the code area, 3012-3141, 3222-3344, 3490-3642 , 3775th position-4332rd position force S intron region.
配列番号： 90によって示される塩基配列の第 1位〜第 2469位がプロモーター領域 (配列番号： 98)であり、第 2792位〜第 2924位、第 3027位〜第 3181位、 3850第 位〜第 4035位、第 4202位〜第 4554位、第 5504位〜第 5684位がコード領域内 のェクソンであり、第 2925位〜第 3026位、第 3182位〜第 3849位、第 4036位〜 第 4201位、第 4555位〜第 5503位力 Sイントロン領域である。  The 1st to 2469th positions of the nucleotide sequence represented by SEQ ID NO: 90 are the promoter region (SEQ ID NO: 98), 2792th to 2924th position, 3027th to 3181th position, 3850th to 3rd position. No. 4035, No. 4202 to No. 4554, No. 5504 to No. 5684 are Exons in the code area, No. 2925 to No. 3026, No. 3182 to No. 3849, No. 4036 to No. 4201 , Positions 4555 to 5503 are S intron regions.
配列番号： 91によって示される塩基配列の第 1位〜第 1242位がプロモーター領域 (配列番号： 99)であり、第 1482位〜第 2347位、第 2452位〜第 3193位がコード領 域内のェクソンであり、第 2348位〜第 2451位がイントロン領域である。  In the nucleotide sequence represented by SEQ ID NO: 91, positions 1 to 1242 are the promoter region (SEQ ID NO: 99), positions 1482 to 2347, and positions 2452 to 3193 are exons in the coding region. 2nd to 2451th positions are intron regions.
配列番号： 92によって示される塩基配列の第 1位〜第 2997位がプロモーター領域 (配列番号： 100)であり、第 3270位〜第 3335位、第 3606位〜第 3757位、第 403 4位〜第 4278位、第 4562位〜第 4690位、第 4797位〜第 5732位、第 5844位〜 第 5971位がコード領域内のヱクソンであり、第 3336位〜第 3605位、第 3758位〜 第 4033位、第 4279位〜第 4561位、第 4691位〜第 4796位、第 5733位〜第 584 3位がイントロン領域である。  The 1st to 2997th positions of the base sequence represented by SEQ ID NO: 92 are the promoter region (SEQ ID NO: 100), the 3270th to 3335th positions, the 3606th position to the 3757th position, the 403th position to the 403th position. No. 4278, No. 4562 to No. 4690, No. 4797 to No. 5732, No. 5844 to No. 5971 are Sae Kwong in the code area, No. 3336 to No. 3605, No. 3758 to No. 4033 , 4279th to 4561th, 4691th to 4796th, 5733th to 584th are the intron regions.
(実施例 5)転写因子群の選抜 実施例 1に示すデータベース力も得られたァノテーシヨン情報を用いて、遺伝子発 現制御に関わる転写因子遺伝子（J. L. Riechmannら SCIENCE VOL 290 15 DECEM BER 2000、岩淵雅榭 '篠崎一雄編、植物ゲノム機能のダイナミズム）と高い相同性を 持つクローンの検索を行った。その結果、 71クローンを特定した。しかしながら、これ らのクローンの中には木繊維形成だけでなぐ榭皮組織等の形成に関わる転写因子 も含まれている。 (Example 5) Selection of transcription factors Transcription factor genes involved in gene expression control (JL Riechmann et al., SCIENCE VOL 290 15 DECEM BER 2000, Kazuo Iwasaki, Kazuo Shinozaki, dynamism of plant genome function, using the annotation information that also obtained the database power shown in Example 1 ) And clones with high homology were searched. As a result, 71 clones were identified. However, these clones also contain transcription factors that are involved in the formation of scab tissue, etc., which is not limited to the formation of wood fibers.
[0184] (実施例 6)木繊維形成に関わる転写因子群の抽出  [0184] (Example 6) Extraction of transcription factor groups involved in wood fiber formation
ァノテーション情報をもとに選抜された 71クローンの中には、木繊維形成だけでなく 、榭皮組織等の形成に関わるものも含まれている。そこで、異なる地上高'種のユー カリ榭幹を用いて、マイクロアレイによる発現解析により、これらの中から木繊維形成 に関わる転写因子群を抽出した。  Among the 71 clones selected based on annotation information, not only wood fiber formation but also those involved in the formation of crust tissue and the like are included. Therefore, transcription factors related to tree fiber formation were extracted from these by eucalyptus trunks of different types of ground heights and by microarray expression analysis.
[0185] 本発明者らは、実施例 1に示した RNA抽出方法に従って、 5年生のカマルドレンシ ス種の樹幹基部と榭幹中央部、グロプルス種の榭幹基部を材料に全 RNAを抽出した 。この際、各植物体の榭皮側 (師部)と木繊維形成組織側 (木部)を用いた。各個体 から得られた全 RNAから、プロメガ社製の PolyATract mRNA Isolation Systemにより、 mRNAを精製した。このようにして得られた合計 4種の mRNAを各々 2種の蛍光色素（c y3.cy5)により標識し、実施例 1に示すオリゴマイクロアレイ解析におけるプローブとし て用い、ハイブリダィゼーシヨンを行った（図 1〜3)。標識を含むハイブリダィゼーショ ンの方法はアジレントテクノロジーより提示の解析プロトコルに従った。スキャンして得 られた画像から蛍光強度を算出し、ロゼッタ社製の解析ソフトウェア (Luminator Ver. 1.0)にて解析し、反復実験全てを統計的信頼度 99.999%にて統合し、 5年生のカマル ドレンシス種の榭幹基部と榭幹中央部、グロプルス種の榭幹基部における選抜した 7 1クローンの発現情報を得た。  [0185] In accordance with the RNA extraction method described in Example 1, the present inventors extracted total RNA from the stem base and trunk center of a 5-year-old Camaldrensis species and the trunk base of a Groplus species. At this time, the husk side (the phloem) and the wood fiber forming tissue side (the xylem) of each plant were used. From the total RNA obtained from each individual, mRNA was purified by PolyATract mRNA Isolation System manufactured by Promega. A total of 4 types of mRNA thus obtained were labeled with 2 types of fluorescent dyes (cy3.cy5), respectively, and used as probes in the oligo microarray analysis shown in Example 1 for hybridization. (Figures 1-3). The hybridization method including the label followed the analysis protocol presented by Agilent Technologies. Fluorescence intensity is calculated from the scanned image, analyzed with Rosetta analysis software (Luminator Ver. 1.0), and all repeated experiments are integrated with a statistical reliability of 99.999%. Information on the expression of 71 clones selected in the trunk base of the drenches species and the center of the trunk, and in the trunk base of the groprus species was obtained.
[0186] ユーカリの木繊維形成に関わる転写因子群は、榭皮側（師部）と比較して木繊維形 成組織側 (木部)で有意に発現が強くなると予想される。選抜した 71クローンの中で、 有意に木繊維形成組織側で強 ヽ発現を示すクローンを選抜した結果、 11クローンを ユーカリの木繊維細胞壁形成に関与している遺伝子群を同定した (表 5〜8)。  [0186] The transcription factor group involved in eucalyptus tree fiber formation is expected to be significantly more strongly expressed on the wood fiber forming tissue side (xylem) than on the crust skin side (phloem). Among the 71 clones selected, clones that significantly expressed strong expression on the side of the wood fiber-forming tissue were selected. As a result, 11 clones identified a group of genes involved in eucalyptus tree fiber cell wall formation (Table 5- 8).
[0187] 本明細書では、同定された cDNA配列を配列番号： 101〜： L 11に示し、それぞれの cDNA力 コードされるアミノ酸配列を配列番号： 112〜122に示す。なお、配列番号[0187] In the present specification, the identified cDNA sequences are shown in SEQ ID NOs: 101 to L11, cDNA force The encoded amino acid sequence is shown in SEQ ID NOs: 112-122. Sequence number
： 112〜 122の!、ずれかに記載のアミノ酸配列からなるタンパク質をコードする DNA は、以下に示す構造的特徴 (ドメイン)を有する。 : A DNA encoding a protein consisting of the amino acid sequence described in any one of 112 to 122 has the following structural features (domains).
配列番号： 112に記載のアミノ酸配列は CBFドメイン、 HAP2ドメイン、 The amino acid sequence set forth in SEQ ID NO: 112 is a CBF domain, a HAP2 domain,
配列番号：113に記載のアミノ酸配列はホメォドメイン、 The amino acid sequence set forth in SEQ ID NO: 113 is a homeodomain,
配列番号： 114に記載のアミノ酸配列は Coprinusjnatingドメイン、 The amino acid sequence set forth in SEQ ID NO: 114 is a Coprinusjnating domain,
配列番号： 115に記載のアミノ酸配列は LIMドメイン、 The amino acid sequence set forth in SEQ ID NO: 115 is a LIM domain,
配列番号：116に記載のアミノ酸配列は bZIPドメイン、 The amino acid sequence set forth in SEQ ID NO: 116 is a bZIP domain,
配列番号： 117に記載のアミノ酸配列は SANTドメイン、 Myb DNA bindingドメイン、 RE B1ド、メイン、 The amino acid sequence set forth in SEQ ID NO: 117 is SANT domain, Myb DNA binding domain, RE B1 domain, main,
配列番号：118に記載のアミノ酸配列は WRKYドメイン、 The amino acid sequence set forth in SEQ ID NO: 118 is the WRKY domain,
配列番号： 119に記載のアミノ酸配列はホメォドメイン、 leucine zipperドメイン、 配列番号： 120に記載のアミノ酸配列は UPF0023ドメイン、 leucine zipperドメイン、 配列番号： 121に記載のアミノ酸配列は SANTドメイン、 Myb DNA bindingドメイン、 RE B1ド、メイン The amino acid sequence described in SEQ ID NO: 119 is homeodomain, leucine zipper domain, the amino acid sequence described in SEQ ID NO: 120 is UPF0023 domain, leucine zipper domain, the amino acid sequence described in SEQ ID NO: 121 is SANT domain, Myb DNA binding domain , RE B1 de, main
配列番号：122に記載のアミノ酸配列は Sinaドメイン。 The amino acid sequence set forth in SEQ ID NO: 122 is the Sina domain.
[表 5] [Table 5]
Figure imgf000087_0001
Figure imgf000087_0001
上記 Sequence IDは上から順に、配列番号 101〜： L I 1に対応する。 The Sequence ID corresponds to SEQ ID NO: 101 to L I 1 in order from the top.
マイクロアレイ実験 5y-cam5 X vs Pは、 5年生ユーカリ 'カマルドレンシス榭幹中央 部の木部と師部を比較した実験を、 5y-caml X vs Pは 5年生ユーカリ 'カマルドレンシ ス基部の木部と師部を比較した実験を、 5y-glo X vs Pは、 5年生ユーカリ 'グロプルス 基部の木部と師部を比較した実験を表す。実験結果は、木繊維形成組織側と榭皮 側を比較して、前者で強い場合は正の倍数で、榭皮側で強い場合は負の倍数で表 している。 The microarray experiment 5y-cam5 X vs P is an experiment comparing the xylem and phloem of the fifth grade eucalyptus 'Camardrensis trunk, 5y-caml X vs P is the fifth grade eucalyptus' camaldrenci 5y-glo X vs P represents an experiment comparing the xylem and phloem of the 5th grade Eucalyptus base. The experimental results are expressed as a positive multiple when the former is strong, and as a negative multiple when strong on the husk side, comparing the wood fiber-forming tissue side and the husk side.
[0190] 11個の遺伝子群は、 5年生のカマルドレンシス種の榭幹基部と中央部における詳 細な発現様式から、以下の（1)と（2)に大別された (表 6、 8)。  [0190] Eleven gene groups were broadly classified into the following (1) and (2) based on the detailed expression patterns in the trunk base and central part of the fifth grade Camaldorensis species (Table 6, 8).
[0191] [表 6] [0191] [Table 6]
Figure imgf000088_0001
Figure imgf000088_0001
[0192] (1) 5個の「ユーカリ属カマルドレンシスの榭幹基部および榭幹中央部において、師 部側と比較して木部側で発現レベルが上昇している遺伝子群」（配列番号： 102〜1 06) [0192] (1) At the five trunk bases and central trunks of the Eucalyptus genus Camaldrensis Gene group whose expression level is increased on the xylem side compared to the xylem side ”(SEQ ID NOs: 102 to 106)
[0193] 5個の遺伝子群には、榭幹基部または榭幹中央部のいずれかにおいて、師部側と 比べ木部側での発現差力 ¾倍以上である遺伝子群 (配列番号： 102、 105、 106)、 榭幹基部または榭幹中央部のいずれかにおいて、師部側と比べ木部側での発現差 が 4倍以上である遺伝子群 (配列番号： 105、 106)が含まれる。  [0193] The five gene groups include a gene group (SEQ ID NO: 102, which has an expression differential power of ¾ times or more on the xylem side compared to the phloem side in either the trunk base or the trunk central part. 105, 106), a group of genes (SEQ ID NO: 105, 106) whose expression difference on the xylem side is 4 times or more compared to the phloem side in either the trunk base or the trunk central part.
[0194] 5個の遺伝子群について、榭幹基部と榭幹中央部の木部側での遺伝子発現を直 接比較した結果、 3個（表 8の垂直変動分類グループ A-2)の「榭幹基部の木部側と 榭幹中央部の木部側での発現が同等な遺伝子群」（配列番号： 104〜106)、 2個（ 表 8の垂直変動分類グループ A-3)の「榭幹基部の木部側と比較して榭幹中央部の 木部側で発現が上昇している遺伝子群」（配列番号： 102、 103)に大別された。より 具体的には、実験によって得られた平均蛍光強度の数値を、榭幹基部 Z中央部また は中央部 Z榭幹基部の式により計算した。有意である各遺伝子の発現情報から、榭 幹基部 Z中央部の式によって得られる数値が 1.1以上かつ Pく =0.001であるものを「榭 幹中央部の木部側と比較して榭幹基部の木部側で発現が上昇している遺伝子群」と 、中央部 Z榭幹基部の式によって得られる数値が 1.1以上かつ Pく =0.001であるものを 「榭幹基部の木部側と比較して榭幹中央部の木部側で発現が上昇している遺伝子 群」と、有意でな 、もしくは平均強度がほぼ同じもの（中央部の木部と基部の木部の 蛍光強度比が 1.0〜1.1倍未満または P〉0.001)を「榭幹基部の木部側と榭幹中央部 の木部側での発現が同等な遺伝子群」と判断し、分類した。  [0194] As a result of a direct comparison of the gene expression at the xylem side of the trunk base and the central trunk of the five gene groups, three groups (vertical variation classification group A-2 in Table 8) "Groups of genes with equivalent expression on the xylem side of the trunk base and the xylem side of the middle of the trunk" (SEQ ID NO: 104-106), 2 (vertical variation classification group A-3 in Table 8) It was roughly classified into “gene groups whose expression is increased on the xylem side of the trunk trunk compared to the xylem side of the trunk base” (SEQ ID NOs: 102 and 103). More specifically, the numerical value of the average fluorescence intensity obtained by the experiment was calculated by the formula of the trunk base Z center or the center Z trunk base. Based on the expression information of each gene that is significant, the value obtained by the formula of 榭 trunk base Z central part is 1.1 or more and P = 0.001 Compared with the xylem side of the trunk base, the numerical value obtained by the formula of the central Z trunk base is 1.1 or more and P = 0.001. Gene group whose expression is increased on the xylem side of the trunk trunk ”is significantly or the same as the average intensity (the fluorescence intensity ratio of the xylem at the center and the xylem at the base is 1.0). Less than -1.1 times or P> 0.001) was classified as a “gene group with similar expression on the xylem side of the trunk base and the xylem side of the trunk trunk”.
[0195] 2個の遺伝子群には、榭幹基部の木部と榭幹中央部の木部での発現差力 ¾倍以上 である遺伝子群 (配列番号： 102、 103)が含まれる。  [0195] The two gene groups include a gene group (SEQ ID NOs: 102 and 103) having an expression differential power of ≥3 times in the xylem of the trunk base and the xylem of the trunk central part.
[0196] (2) 5個（表 8の垂直変動分類グループ G)の「ユーカリ属カマルドレンシスの榭幹中 央部において、師部側と比較して木部側で発現レベルが上昇し、榭幹基部において[0196] (2) In 5 (vertical variation classification group G in Table 8), the expression level increased in the xylem side compared to the phloem side in the middle of the trunk of Eucalyptus genus Camaldrensis, At the trunk base
、師部側と木部側で発現レベルが同等な遺伝子群」（配列番号： 101、 107、 109〜, Gene groups with similar expression levels on the phloem side and xylem side ”(SEQ ID NOs: 101, 107, 109-
111) 111)
[0197] 5個の遺伝子群には、榭幹中央部において、師部側と比べて木部側での発現差が 1.5倍以上である遺伝子群 (配列番号： 107、 109〜111)、榭幹中央部において、師 部側と比べて木部側での発現差力 ^倍以上である遺伝子群 (配列番号： 107、 109) が含まれる。 [0197] The five gene groups have a gene group (SEQ ID NOs: 107, 109 to 111) in which the expression difference on the xylem side is 1.5 times or more in the central trunk portion compared to the phloem side, 榭In the center of the trunk, teacher A gene group (SEQ ID NOs: 107 and 109) having an expression differential power of ^ times or more on the xylem side compared to the xylem side is included.
[0198] 11個の遺伝子群は、 5年生のカマルドレンシス種の榭幹基部と榭幹中央部、 5年生 のグロプルス種の榭幹基部における詳細な発現様式から、以下の（1)〜（3)に大別 された (表 7、 8)。  [0198] Eleven gene groups are expressed in the following (1) to (1) from the detailed expression patterns in the trunk base and central trunk of the fifth grade Camaldorensis species and the trunk base of the fifth grade Groplus species. They were broadly classified into 3) (Tables 7 and 8).
[0199] [表 7]  [0199] [Table 7]
Figure imgf000090_0001
Figure imgf000090_0001
[0200] [表 8] 分類グループ 分類グループ[0200] [Table 8] Classification group Classification group
Sequence ID 一次配列名 Sequence ID Primary sequence name
(垂直変動） (樹種間変動）  (Vertical fluctuation) (Variation between trees)
77629 Stem.001.005.1.CL7.cl.791.Contig1 G I  77629 Stem.001.005.1.CL7.cl.791.Contig1 G I
76469 Stem.001.005.1.CL.24.cl.2440.Contig1 A - 3 E  76469 Stem.001.005.1.CL.24.cl.2440.Contig1 A-3 E
73643 Stem— 001— 0001 69B05 A - 3 A-2 73643 Stem— 001— 0001 69B05 A-3 A-2
72727 Stem_001 _005 J .CL2.cl.221.Contig2 A - 2 C 72727 Stem_001 _005 J .CL2.cl.221.Contig2 A-2 C
75229 Stem.005_000008H07 A-2 A - 3 75229 Stem.005_000008H07 A-2 A-3
73384 Stem_005_000023F02 A-2 C 73384 Stem_005_000023F02 A-2 C
72062 Stem.001.005_1.CL14.cl.1422.Contig1 G I  72062 Stem.001.005_1.CL14.cl.1422.Contig1 G I
73448 Stem— 001-005— 1.CL20.cl.2009.Contig1 I I  73448 Stem— 001-005— 1.CL20.cl.2009.Contig1 I I
73187 Stem— 005— 000049F1 1 G I  73187 Stem— 005— 000049F1 1 G I
74387 Stem— 001— 000293H 12 G I  74387 Stem— 001— 000293H 12 G I
75267 Stem_001_005_1.CL6.cl.624.Contig1 G I  75267 Stem_001_005_1.CL6.cl.624.Contig1 G I
[0201] (1) 2個の「ユーカリ属カマルドレンシスおよびユーカリ属グロブルスの榭幹において 、師部側と比較して木部側で発現レベルが上昇している遺伝子群」（配列番号： 103 、 105) [0201] (1) Two “groups of genes whose expression levels are increased on the xylem side compared to the phloem side in the trunks of Eucalyptus genus Camaldrensis and Eucalyptus globules” (SEQ ID NO: 103 , 105)
[0202] 2個の遺伝子群には、ユーカリ属カマルドレンシスおよびユーカリ属グロブルスの榭 幹の 、ずれかにお 、て、師部側と比べ木部側での発現差が 10倍以上である遺伝子 群 (配列番号： 105)が含まれる。  [0202] In the two gene groups, eucalyptus chamaldrensis and Eucalyptus globulus trunks are more than 10 times more differentially expressed on the xylem side than on the phloem side The gene group (SEQ ID NO: 105) is included.
[0203] 2個の遺伝子について、ユーカリ属カマルドレンシスの榭幹基部または中央部の木 部側とユーカリ属グロブルスの榭幹基部の木部側での遺伝子発現を直接比較した結 果、 1個（表 8の榭種間変動分類グループ A-2)の「ユーカリ属カマルドレンシスの榭幹 の木部側とユーカリ属グロブルスの樹幹の木部側での発現が同等な遺伝子」（配列 番号： 103)、 1個（表 8の榭種間変動分類グループ A-3)の「ユーカリ属グロブルスの 榭幹の木部側と比較してユーカリ属カマルドレンシスの樹幹の木部側で発現レベル が減少している遺伝子」（配列番号： 105)に大別された。より具体的には、実験によ つて得られた平均蛍光強度の数値を、ユーカリ属カマルドレンシスの榭幹 (基部また は中央部）の木部 Zユーカリ属グロブルスの榭幹 (基部）の木部の式またはユーカリ 属グロブルスの榭幹 (基部）の木部 Zユーカリ属カマルドレンシスの榭幹 (基部または 中央部）の木部の式により計算した。各遺伝子の発現情報から、ユーカリ属カマルド レンシス Zユーカリ属グロブルスの樹幹の木部の式によって得られる数値が l.i以上 かつ P〈=0.001であるものを「ユーカリ属グロブルスの榭幹の木部側と比較してユー力 リ属カマルドレンシスの樹幹の木部側で発現が上昇している遺伝子群」と、ユーカリ 属グロブルスの榭幹 Zユーカリ属カマルドレンシスの木部の式によって得られる数値 力 S1.1以上かつ P〈=0.001であるものを「ユーカリ属カマルドレンシスの樹幹の木部側と 比較してユーカリ属グロブルスの樹幹の木部側で発現が上昇して 、る遺伝子群」と、 有意でないもしくは平均強度がほぼ同じもの（ユーカリ属グロブルスの樹幹の木部に 比べてユーカリ属カマルドレンシスの樹幹の木部の蛍光強度比が 1.0〜 1.1倍未満ま たは P〉0.001)を「ユーカリ属カマルドレンシスの榭幹の木部側とユーカリ属グロブルス の榭幹の木部側での発現が同等な遺伝子群」と判断し、分類した。 [0203] As a result of direct comparison of gene expression on the xylem side of the stem base or central part of Eucalyptus chamaldrensis and the xylem side of the stem base of Eucalyptus globulus for two genes, one (Gene variation classification group A-2 in Table 8) “A gene whose expression on the xylem side of Eucalyptus chamaldrensis is equivalent to that on the xylem side of Eucalyptus globulus” (SEQ ID NO: 103), 1 (in the varietal variation classification group A-3 in Table 8), the expression level is higher on the xylem side of the Eucalyptus chamaldrensis trunk than on the xylem side of the Eucalyptus globulus trunk. It was roughly divided into “decreasing genes” (SEQ ID NO: 105). More specifically, the value of the average fluorescence intensity obtained from the experiment was calculated based on the trunk of the Eucalyptus chamaldrensis trunk (base or center) Z the trunk of the Eucalyptus globulus (base). It was calculated by the formula of the part or the xylem of the Eucalyptus globulus trunk (base) Z of the trunk of the eucalyptus chamaldrensis (base or middle part). From the expression information of each gene, Rensis Z Eucalyptus globulus tree part formula obtained by the formula of li or higher and P <= 0.001 compared to the eucalyptus globulus trunk trunk side, `` A gene group whose expression is increased on the xylem side of the tree trunk '' and the numerical force obtained from the eucalyptus globulus trunk Z-eucalyptus chamaldrensis xylem formula S1.1 or higher and P <= 0.001 Some genes are not significant or have almost the same average intensity as "a group of genes whose expression increases on the xylem side of the Eucalyptus globulus tree compared to the xylem side of the Eucalyptus genus Camaldrensis" (The fluorescence intensity ratio of the tree part of Eucalyptus genus Camaldrensis compared to the tree part of Eucalyptus globulis 1.0 to 1.1 times or P> 0.001) Kibe side and Expression in xylem side 榭幹 of Kari genus globulus is determined that equivalent genes "were classified.
[0204] (2) 2個（表 8の榭種間変動分類グループ C)の「ユーカリ属カマルドレンシスの榭幹 において、師部側と比較して木部側で発現レベルが上昇し、ユーカリ属グロブルスの 榭幹において、師部側と比較して木部側で発現レベルが減少している遺伝子群」（ 配列番号： 104、 106)  [0204] (2) In the trunk of Eucalyptus genus Camaldrensis, the expression level increased on the xylem side compared to the phloem side, and two eucalyptus varieties (Table 8). In the trunk of the genus Globulus, the gene group whose expression level is reduced on the xylem side compared to the phloem side "(SEQ ID NOs: 104, 106)
[0205] 2個の遺伝子群には、ユーカリ属カマルドレンシスの榭幹において、師部側と比べ 木部側での発現差が 4倍以上、またはユーカリ属グロブルスの榭幹において、木部側 と比べ師部側での発現差が 4倍以上である遺伝子 (配列番号：106)が含まれる。  [0205] In the two gene groups, the expression difference on the xylem side was more than 4-fold in the trunk of Eucalyptus genus Camaldrensis, or the xylem side in the trunk of Eucalyptus globules The gene (SEQ ID NO: 106) whose expression difference on the phloem side is 4 times or more is included.
[0206] (3) 1個（表 8の榭種間変動分類グループ E)の「ユーカリ属カマルドレンシスの榭幹 において、師部側と比較して木部側で発現レベルが上昇し、ユーカリ属グロブルスの 榭幹において、師部側と木部側で発現レベルが同等な遺伝子群」（配列番号：102) [0206] (3) In the trunk of Eucalyptus genus Camaldrensis, the expression level increased on the xylem side compared to the phloem side, and one eucalyptus In the trunk of the genus Globulus, genes with similar expression levels on the phloem side and xylem side ”(SEQ ID NO: 102)
[0207] (実施例 7)木繊維形成に関わる転写因子群のゲノムクローンとプロモーター DNAの 取得 [Example 7] Acquisition of genomic clones and promoter DNA of transcription factors involved in wood fiber formation
(1)ユーカリ BACゲノムライブラリーのスクリーニング  (1) Eucalyptus BAC genomic library screening
ユーカリ BACゲノムライブラリーのスクリーニングは、実施例 4に示す方法で行った 。 PCRによる 3Dスクリーニング用の DNAプールを铸型に、 cDNA配列情報を基に合 成した配列番号： 123〜 144のオリゴヌクレオチドプライマー（配列番号： 123〜144 の奇数番号はフォワードプライマー、偶数番号はリバースプライマー）を用い、 PCR法 により各遺伝子のゲノムクローンのスクリーニングを行った。 [0208] BACクローンのインサート配列は、実施例 1に示す方法で決定した。得られた塩基 配列を用い、 cDNA配列をもとに各遺伝子のゲノム領域とプロモーター領域を決定し た（配列番号： 101〜： L 11に記載の cDNA配列をもとに決定されたゲノム配列を、それ ぞれ、配列番号： 145〜155に示す)。また、翻訳開始コドン ATG (アミノ酸ではメチォ ニンをコード）を含む全長の cDNA配列をもとにプロモーター領域を決定した。決定さ れたプロモーター配列を配列番号： 156〜166に示した。具体的には、全長 cDNA の 5 '末端塩基から上流の配列を、遺伝情報処理ソフトウェア「GENETYX」（ソフトゥェ ァ開発株式会社）により解析し、 TATA-box配列を決定した。高等真核生物のプロモ 一ター領域には、転写開始点より通常約 10〜25塩基上流側に TATA-box配列がある ことがよく知られている。決定された TATA-boxの位置から全長 CDNA5 '末端塩基ま でが近接している (約 lOObp程度)場合、 cDNAはほぼ完全長であり、その末端塩基 上流側がプロモーター領域であると判断した。プロモーターの長さは、モデル植物で 知られている最大の長さ（約 3kb)を参考に、 cDNA5'末端塩基から上流側最大 3kb までとした。 The Eucalyptus BAC genomic library was screened by the method shown in Example 4. Oligonucleotide primer of SEQ ID NO: 123-144 synthesized based on cDNA sequence information in a DNA pool for 3D screening by PCR, based on cDNA sequence information (odd number of SEQ ID NO: 123-144 is forward primer, even number is reverse) Using primers, the genomic clones of each gene were screened by PCR. [0208] The insert sequence of the BAC clone was determined by the method shown in Example 1. Using the obtained base sequence, the genomic region and the promoter region of each gene were determined based on the cDNA sequence (SEQ ID NO: 101 to: The genomic sequence determined based on the cDNA sequence described in L11 was determined. , Respectively, as shown in SEQ ID NOs: 145-155). The promoter region was determined based on the full-length cDNA sequence including the translation initiation codon ATG (which encodes methionine in amino acids). The determined promoter sequence is shown in SEQ ID NOs: 156-166. Specifically, the sequence upstream from the 5 'terminal base of the full-length cDNA was analyzed by genetic information processing software "GENETYX" (Software Development Co., Ltd.), and the TATA-box sequence was determined. It is well known that the promoter region of higher eukaryotes has a TATA-box sequence usually about 10 to 25 bases upstream from the transcription start site. When the position of the determined TATA-box was close to the full-length CDNA 5 ′ terminal base (about lOObp), it was judged that the cDNA was almost full length and that the upstream side of the terminal base was the promoter region. The length of the promoter was determined from the 5 ′ end of the cDNA to a maximum of 3 kb upstream from the maximum length (about 3 kb) known in model plants.
[0209] 配列番号： 101によって示される塩基配列では、第 87位〜第 662位がアミノ酸コー ド領域 (配列番号：112)である。  [0209] In the nucleotide sequence represented by SEQ ID NO: 101, the 87th to 662nd positions are the amino acid code region (SEQ ID NO: 112).
配列番号： 102によって示される塩基配列では、第 262位〜第 1188位がアミノ酸コ ード領域 (配列番号： 113)である。  In the base sequence represented by SEQ ID NO: 102, positions 262 to 1188 are the amino acid code region (SEQ ID NO: 113).
配列番号： 103によって示される塩基配列では、第 257位〜第 1549位がアミノ酸コ ード領域 (配列番号： 114)である。  In the nucleotide sequence represented by SEQ ID NO: 103, the 257th to 1549th positions are the amino acid code region (SEQ ID NO: 114).
配列番号： 104によって示される塩基配列では、第 159位〜第 722位がアミノ酸コ ード領域 (配列番号：115)である。  In the base sequence represented by SEQ ID NO: 104, positions 159 to 722 are the amino acid code region (SEQ ID NO: 115).
配列番号： 105によって示される塩基配列では、第 101位〜第 1444位がアミノ酸コ ード領域 (配列番号：116)である。  In the base sequence represented by SEQ ID NO: 105, positions 101 to 1444 are the amino acid code region (SEQ ID NO: 116).
配列番号： 106によって示される塩基配列では、第 163位〜第 927位がアミノ酸コ ード領域 (配列番号： 117)である。  In the base sequence represented by SEQ ID NO: 106, the 163rd to 927th positions are the amino acid code region (SEQ ID NO: 117).
配列番号： 107によって示される塩基配列では、第 48位〜第 2300位がアミノ酸コ ード領域 (配列番号：118)である。 配列番号： 108によって示される塩基配列では、第 23位〜第 778位がアミノ酸コー ド領域 (配列番号：119)である。 In the nucleotide sequence represented by SEQ ID NO: 107, the 48th to 2300th positions are the amino acid code region (SEQ ID NO: 118). In the base sequence represented by SEQ ID NO: 108, the 23rd to 778th positions are the amino acid code region (SEQ ID NO: 119).
配列番号： 109によって示される塩基配列では、第 40位〜第 1101位がアミノ酸コ ード領域 (配列番号： 120)である。  In the nucleotide sequence represented by SEQ ID NO: 109, positions 40 to 1101 are the amino acid code region (SEQ ID NO: 120).
配列番号： 110によって示される塩基配列では、第 128位〜第 790位がアミノ酸コ ード領域 (配列番号： 121)である。  In the base sequence represented by SEQ ID NO: 110, the 128th to 790th positions are the amino acid code region (SEQ ID NO: 121).
配列番号： 111によって示される塩基配列では、第 269位〜第 1234位がアミノ酸コ ード領域 (配列番号： 122)である。  In the base sequence represented by SEQ ID NO: 111, positions 269 to 1234 are the amino acid code region (SEQ ID NO: 122).
配列番号： 145によって示される塩基配列の第 1位〜第 2680位がプロモーター領 域 (配列番号： 156)であり、第 2767位〜第 2796位、第 2884位〜第 2949位、第 2 984位〜第 3142位、第 3265位〜第 3585位がコード領域内のエタソンであり、第 2 797位〜第 2883位、第 2950位〜第 2983位、第 3143位〜第 3264位、がイントロ ン領域である。  The 1st to 2680th positions of the nucleotide sequence represented by SEQ ID NO: 145 are the promoter region (SEQ ID NO: 156), the 2767th to 2796th positions, the 2884th to 2949th positions, and the 2nd 984th position. -3142, 3265-3585 are etasons in the code area, 29797-2883, 2950-2983, 3143-3264, intron It is.
配列番号： 146によって示される塩基配列の第 1位〜第 3000位がプロモーター領 域 (配列番号： 157)であり、第 3262位〜第 3606位、第 3920位〜第 4040位、第 5 436位〜第 5626位、第 5829位〜第 5960位、第 6246位〜第 6383位、力 Sコード領 域内のェクソンであり、第 3607位〜第 3919位、第 4041位〜第 5435位、第 5627 位〜第 5828位、第 5961位〜第 6245位力 Sイントロン領域である。  The 1st to 3000th positions of the nucleotide sequence represented by SEQ ID NO: 146 are the promoter region (SEQ ID NO: 157), positions 3262 to 3606, positions 3920 to 4040, and positions 5436. -5626th, 5829th-5960th, 6246th-6383th, Ekson in the force S code area, 3607th-3919th, 4041st-5435th, 5627th ~ No. 5828, No. 5961 ~ No. 6245 position S intron region.
配列番号： 147によって示される塩基配列の第 1位〜第 1569位がプロモーター領 域 (配列番号： 158)であり、第 1826位〜第 2533位、第 4847位〜第 4967位、第 5 439位〜第 5632位、第 5709位〜第 5840位、第 5936位〜第 6072位、第 6158位 がコード領域内のエタソンであり、第 2534位〜第 4846位、第 4968位〜第 5438位 、第 5633位〜第 5708位、第 5841位〜第 5935位、第 6073位〜第 6157位がイン トロン領域である。  The 1st to 1569th positions of the nucleotide sequence represented by SEQ ID NO: 147 are the promoter region (SEQ ID NO: 158), the 1826th to 2533th positions, the 4847th to 4967th positions, and the 5439th position. -5632, 5709-5840, 5936-6072, 6158 are Etason in the code region, 2534-4846, 4968-5438, Intron regions are 5633 to 5708, 5841 to 5935, and 6073 to 6157.
配列番号： 148によって示される塩基配列の第 1位〜第 2979位がプロモーター領 域 (配列番号： 159)であり、第 3241位〜第 3375位、第 3967位〜第 4063位、、第 4159位〜第 4202位、第 4293位〜第 4282位、第 4539位〜第 4736位がコード領 域内のェクソンであり、第 3376位〜第 3966位、第 4064位〜第 4158位、第 4203 位〜第 4292位、第 4283位〜第 4538位力 Sイントロン領域である。 The first to 2979th position of the nucleotide sequence represented by SEQ ID NO: 148 is the promoter region (SEQ ID NO: 159), 3241th to 3375th position, 3967th to 4063th position, 4159th position. -No. 4202, No. 4293-No. 4282, No. 4539-No. 4736 are Eksons in the code area, No. 3376-No. 3966, No. 4064-No. 4158, No. 4203 Positions-4292, 4283-4538 positions S intron region.
配列番号： 149によって示される塩基配列の第 1位〜第 1288位がプロモーター領 域 (配列番号： 160)であり、第 1409位〜第 1964位、第 3755位〜第 3959位、第 4 510位〜第 4585位、第 4564位〜第 4809位、第 5066位〜第 5446位力コード領 域内のェクソンであり、第 1965位〜第 3754位、第 3960位〜第 4509位、第 4586 位〜第 4563位、第 4810位〜第 5065位がイントロン領域である。  The 1st to 1288th positions of the nucleotide sequence represented by SEQ ID NO: 149 are the promoter region (SEQ ID NO: 160), 1409th to 1964th position, 3755th to 3959th position, 4th 510th position -4585th, 4564th-4809th, 5066th-5446th Exon in the power code area, 1965-3754, 3960-4509, 4586-4586 The 4563rd and 4810th to 5065th positions are intron regions.
配列番号： 150によって示される塩基配列の第 1位〜第 1536位がプロモーター領 域 (配列番号： 161)であり、第 1723位〜第 1985位、第 2217位〜第 2718位がコー ド領域内のェクソンであり、第 1986位〜第 2216位がイントロン領域である。  In the nucleotide sequence represented by SEQ ID NO: 150, positions 1 to 1536 are the promoter region (SEQ ID NO: 161), positions 1723 to 1985, and positions 2217 to 2718 are in the code region. No. 1986 to No. 2216 are intron regions.
配列番号：151によって示される塩基配列の第 1位〜第 1658位がプロモーター領 域 (配列番号： 162)であり、第 1706位〜第 2167位、第 2407位〜第 2589位、第 2 767位〜第 3647位、第 3747位〜第 3905位、第 4108位〜第 4675位がコード領 域内のェクソンであり、第 2168位〜第 2406位、第 2590位〜第 2766位、第 3648 位〜第 3746位、第 3906位〜第 4107位がイントロン領域である。  The 1st to 1658th positions of the nucleotide sequence represented by SEQ ID NO: 151 are the promoter region (SEQ ID NO: 162), the 1706th to 2167th positions, the 2407th to 2589th positions, the 2767th position. -No. 3647, No. 3747-No. 3905, No. 4108-No. 4675 are Eksons in the code area, No. 2168-No. 2406, No. 2590-No. 2766, No. 3648-No. The intron region is at positions 3746 and 3906-4107.
配列番号： 152によって示される塩基配列の第 1位〜第 1979位がプロモーター領 域 (配列番号： 163)であり、第 2002位〜第 2427位、第 2714位〜第 2793位、第 2 984位〜第 3233位がコード領域内のヱクソンであり、第 2428位〜第 2713位、第 2 794位〜第 2983位がイントロン領域である。  The 1st to 1979th positions of the nucleotide sequence represented by SEQ ID NO: 152 are the promoter region (SEQ ID NO: 163), positions 2002 to 2427, positions 2714 to 2793, positions 2984. ~ Position 3233 is Xun Xun in the coding region, Positions 2428 to 2713 and Positions 2794 to 2983 are intron regions.
配列番号： 153によって示される塩基配列の第 1位〜第 596位がプロモーター領域 (配列番号： 164)であり、第 637位〜第 770位、第 1143位〜第 1272位、第 1393 位〜第 1593位、第 2042位〜第 2209位、第 2939位〜第 3367位がコード領域内 のェクソンであり、第 771位〜第 1142位、第 1273位〜第 1392位、第 1594位〜第 2041位、第 2210位〜第 2938位がイントロン領域である。  The 1st to 596th positions of the nucleotide sequence represented by SEQ ID NO: 153 are the promoter region (SEQ ID NO: 164), positions 637 to 770, positions 1143 to 1272, positions 1393 to 1 No. 1593, No. 2042 to No. 2209, No. 2939 to No. 3367 are exons in the code region, No. 771 to No. 1142, No. 1273 to No. 1392, No. 1594 to No. 2041 , Positions 2210 to 2938 are intron regions.
配列番号： 154によって示される塩基配列の第 1位〜第 2973位がプロモーター領 域 (配列番号： 165)であり、第 3101位〜第 3221位、第 3339位〜第 3468位、第 4 324位〜第 4735位がコード領域内のエタソンであり、第 3222位〜第 3338位、第 3 469位〜第 4323位がイントロン領域である。  In the nucleotide sequence represented by SEQ ID NO: 154, positions 1 to 2973 are the promoter region (SEQ ID NO: 165), positions 3101 to 3221, positions 3339 to 3468, and positions 4324. ˜4735 is the etason in the coding region, 3222 to 3338, and 3469 to 4323 is the intron region.
配列番号： 155によって示される塩基配列の第 1位〜第 1783位がプロモーター領 域 (配列番号： 166)であり、第 2052位〜第 2270位、第 3339位〜第 3725位、第 4 266位〜第 4625位がコード領域内のヱクソンであり、第 2271位〜第 3338位、第 3 726位〜第 4265位がイントロン領域である。 Sequence number 1 to position 1783 of the nucleotide sequence represented by SEQ ID NO: 155 is the promoter region Region (SEQ ID NO: 166), positions 2052 to 2270, positions 3339 to 3725, positions 4266 to 4625 are Xeon in the coding region, positions 2271 to 3338 , Positions 3726 to 4265 are intron regions.
[0210] (実施例 8)木部特異的プロモーター活性の解析 [0210] (Example 8) Analysis of xylem specific promoter activity
(1)活性解析用コンストラクトの構築  (1) Construction of activity analysis construct
実施例 3と 6に示した木部形成組織で発現している遺伝子 (配列番号： 1〜34、 101 〜： L 11)は、それらの各々のプロモーターによって木部での発現が制御されている。 従って、各プロモーター領域は、任意の遺伝子の木部での発現制御に当然用いるこ とができる。そこで、それらのプロモーター下流にレポーター遺伝子をつなぎ、植物に 導入し木部での発現活性を確認した。本実施例では、セルロース合成酵素遺伝子（ 配列番号： 3)のプロモーター領域 (配列番号： 93)の活性を、パーティクルガン法に より解析した例を示す。  The genes (SEQ ID NOs: 1-34, 101-: L11) expressed in the xylem forming tissues shown in Examples 3 and 6 are regulated in the xylem by their respective promoters. . Therefore, each promoter region can naturally be used for expression control in the xylem of an arbitrary gene. Therefore, reporter genes were connected downstream of these promoters and introduced into plants to confirm expression activity in xylem. In this example, the activity of the promoter region (SEQ ID NO: 93) of the cellulose synthase gene (SEQ ID NO: 3) was analyzed by the particle gun method.
木部特異的プロモーターとしての活性を確認するため、セルロース合成酵素遺伝 子（配列番号： 3)のプロモーター領域下流にレポーターとして GUS遺伝子をつなげ ユーカリへ導入した。具体的には、配列番号: 85に記載のゲノム配列情報を基に合 成した配列番号： 167、 168のオリゴヌクレオチドプライマーを用い、 BACクローン DN Aを铸型に PCR法により 5'非翻訳領域を含むプロモーター領域を増幅し、平滑末端 化後 35Sプロモーターを除いたバイナリーベクター pBI121ベクター（Bevan, M., Nucle ic Acids Res. 12, 8711-8721, 1984)の GUS遺伝子上流へサブクローユングした。  In order to confirm the activity as a xylem specific promoter, the GUS gene was connected as a reporter downstream of the promoter region of the cellulose synthase gene (SEQ ID NO: 3) and introduced into Eucalyptus. Specifically, using the oligonucleotide primer of SEQ ID NO: 167, 168 synthesized based on the genome sequence information described in SEQ ID NO: 85, the BAC clone DNA is in a saddle shape and 5 ′ untranslated region by PCR. The promoter region containing was amplified, and after blunting, it was subcloned upstream of the GUS gene of the binary vector pBI121 vector (Bevan, M., Nucleic Acids Res. 12, 8711-8721, 1984) excluding the 35S promoter.
[0211] (2)木部特異的プロモーター活性の解析 [0211] (2) Analysis of xylem specific promoter activity
プロモーター活性の解析として、前述の導入用プラスミドを木部形成組織にパーテ ィクルガンで導入し、 GUS遺伝子の活性を解析した。旺盛に生育中の 5年生ユーカリ 榭幹の樹皮を剥 、だ面に露出して 、る木部形成組織（図 4A)力も木部ブロック（図 4 B)を切除し、パーティクルガン導入用ターゲットとした。導入用プラスミドの金粒子へ のコーティングは、後述のように行った。金粒子 50 1にプラスミド DNAを 5 1 (0.5 g/ μ 1)と 2.5Μ CaCa2を 50 μ 1カ卩えすぐに混合し、次に 0.1Mスペルミジンを 20 μ 1加えさら に混合後、遠心機で金粒子を沈殿させた。 100%エタノールで洗浄した後、 100%ェタノ ール 60 μ 1に懸濁し、 1回分 (5-10 μ 1)ずつマクロキャリアーに分注し乾燥させて用い た。導入条件は、真空度 :27-28 inches Hg、ラプチヤーディスク： 1800psi、金粒子径： 1.6 m、試料距離: 4-8cmで行った。木部形成組織ブロックに導入処理 (ボンバード メント）後、 25°C暗所にて一晩培養し、組織ィ匕学的方法 (X- glu溶液にて 37°C24時 間インキュベート、 Kosugi et al.， 1990, Plant Sci., 70:133-140)にて GUS活性（青い 発色）の検出を行った。その結果、図 4B〜Dに示すように、セルロース合成酵素遺伝 子プロモーターによる GUS遺伝子の発現を示す青いスポットが観察された。従って、 本プロモーターは、マイクロアレイ実験の結果の通り、木部形成部位で発現する活性 を持つことが示された。 As an analysis of promoter activity, the above-described plasmid for introduction was introduced into a xylem-forming tissue with a particle gun, and the activity of the GUS gene was analyzed. 5th year eucalyptus growing vigorously Peeling the bark of the trunk and exposing it to the other side, the xylem formation tissue (Fig. 4A) force also cuts the xylem block (Fig. 4B), and the target for particle gun introduction did. The introduction plasmid was coated on gold particles as described below. Immediately mix 50 μl of plasmid DNA with 51 (0.5 g / μ1) and 50 μl of 2.5 2 CaCa2, and add 20 μl of 0.1M spermidine. To precipitate gold particles. After washing with 100% ethanol, suspend in 100% ethanol 60 μ1, dispense into a macrocarrier once (5-10 μ1), dry and use. It was. The introduction conditions were as follows: degree of vacuum: 27-28 inches Hg, rupture disk: 1800 psi, gold particle size: 1.6 m, sample distance: 4-8 cm. After introduction treatment (bombardment) into xylem-forming tissue blocks, culture overnight in a dark place at 25 ° C, and histological method (incubate with X-glu solution at 37 ° C for 24 hours, Kosugi et al. 1990, Plant Sci., 70: 133-140), GUS activity (blue color development) was detected. As a result, as shown in FIGS. 4B to 4D, blue spots indicating the expression of the GUS gene by the cellulose synthase gene promoter were observed. Therefore, this promoter was shown to have an activity expressed at the xylem formation site as a result of the microarray experiment.
この例からも示されるように、マイクロアレイ実験により木部に発現が認められた配 列番号： 1〜34、 101〜： L 11に記載の遺伝子のプロモーター配列は、任意の遺伝子 の木部での発現制御に用いることができる。  As shown in this example, the promoter sequence of the gene described in SEQ ID NOs: 1-34, 101-: L11, in which expression was observed in the xylem by the microarray experiment, is the xylem in any gene. It can be used for expression control.
[0212] (実施例 9)木繊維形成遺伝子プロモーターと転写因子との相互作用解析 [0212] (Example 9) Analysis of interaction between promoter and transcription factor of wood fiber formation gene
転写因子は、その制御下にある遺伝子のプロモーター領域に結合することで、発 現の on/off^制御している（J. L. Riechmannら SCIENCE VOL 290 15 DECEMBER 20 00、岩淵雅榭 '篠崎一雄編、植物ゲノム機能のダイナミズム)。したがって、遺伝子の プロモーター領域との相互作用（結合)の有無を解析することで、制御'被制御の関 係が分かる。  Transcription factor is controlled on / off ^ by binding to the promoter region of the gene under its control (JL Riechmann et al., SCIENCE VOL 290 15 DECEMBER 2000, Masaaki Iwabuchi, Kazuo Shinozaki, Plant genome function dynamism). Therefore, by analyzing the presence or absence of interaction (binding) with the promoter region of a gene, the relationship between control and control can be understood.
[0213] そこで、木細胞壁形成を制御する配列番号： 101〜： L 11 (配列番号： 112〜122) に記載の転写因子と、配列番号 1〜34 (配列番号 35〜68)に記載の木部細胞壁形 成に関わる遺伝子のプロモーターとの相互作用を解析した。本実施例では、例として 配列番号： 117の MYB転写因子タンパク質と配列番号： 119のホメォドメイン (HD-ZIP )転写因子タンパク質につ 、て、セルロース合成に関わる遺伝子（CesA)のプロモー ター領域 (配列番号： 93)とリグニン合成に関わる遺伝子（CAD、 OMT、 C4H)のプロ モーター（それぞれ配列番号： 96、 97、 99)との相互作用の結果を示す。  [0213] Therefore, the transcription factor described in SEQ ID NO: 101-: L 11 (SEQ ID NO: 112-122) that controls the formation of tree cell walls and the tree described in SEQ ID NO: 1-34 (SEQ ID NO: 35-68) We analyzed the interaction of genes involved in cell wall formation with promoters. In this example, the MYB transcription factor protein of SEQ ID NO: 117 and the homeodomain (HD-ZIP) transcription factor protein of SEQ ID NO: 119 are used as promoter regions (sequences of the gene involved in cellulose synthesis (CesA). No. 93) shows the result of interaction between the lignin synthesis gene (CAD, OMT, C4H) promoter (SEQ ID NO: 96, 97, 99, respectively).
[0214] (1)無細胞翻訳系によるタンパク質発現と精製  [0214] (1) Protein expression and purification by cell-free translation system
無細胞翻訳系により MYB転写因子タンパクを発現させるため、配列番号： 169、 17 0に記載のプライマーを用いて、 MYBの cDNA (配列番号： 106)を铸型に PCRした。 増幅した断片を制限酵素 Ndelと smalで処理し、 pIVEX 1.3ベクター (Roche)に挿入し、 無細胞翻訳系（小麦胚芽タンパク質合成系キット RTS100;Roche)を用いて、 24°C、 24 時間反応させた。この反応液 lulを SDS-PAGEで泳動し、 ant His抗体を用いたウェス タンブロッテイングにより、 目的タンパク質の発現を確認した。 In order to express the MYB transcription factor protein by a cell-free translation system, the MYB cDNA (SEQ ID NO: 106) was PCR-enhanced in a saddle shape using the primers shown in SEQ ID NOs: 169 and 170. Treat the amplified fragment with restriction enzymes Ndel and smal and insert it into pIVEX 1.3 vector (Roche) The reaction was performed at 24 ° C. for 24 hours using a cell-free translation system (wheat germ protein synthesis system kit RTS100; Roche). The reaction mixture (lul) was electrophoresed on SDS-PAGE, and expression of the target protein was confirmed by Western blotting using an ant His antibody.
[0215] (2)大腸菌によるタンパク質発現と精製 [0215] (2) Protein expression and purification by E. coli
大腸菌により HD-ZIP転写因子タンパクを発現させるため、配列番号： 171、 172に 記載のプライマーを用いて、 HD-ZIPの cDNA (配列番号： 108)を铸型に PCRした。 増幅された DNA断片を制限酵素 EcoRVと Sailで処理後、 pET32aベクター (Novagen) に挿入し、大腸菌 BL21系統にトランスフォーメーションした。タンパク質を発現させる 場合は、 LB培地に植菌し OD =0.8になるまで 37°Cで培養し、 ImM IPTGをカ卩ぇ 25°C  In order to express the HD-ZIP transcription factor protein in E. coli, HD-ZIP cDNA (SEQ ID NO: 108) was subjected to PCR using the primers described in SEQ ID NOs: 171 and 172. The amplified DNA fragment was treated with restriction enzymes EcoRV and Sail, inserted into pET32a vector (Novagen), and transformed into E. coli BL21 strain. For protein expression, inoculate in LB medium and incubate at 37 ° C until OD = 0.8, and ImM IPTG at 25 ° C
600  600
、 4時間で培養した。集菌後、 HisTrap HP (GE Healthcare)を用いて精製を行い、 50m Mイミダゾールで洗浄し、 400mMイミダゾールで溶出した。この精製タンパク質を HiT rap Desalting (GE Healthcare)を用いて ft塩した。  Incubated for 4 hours. After collection, purification was performed using HisTrap HP (GE Healthcare), washed with 50 mM imidazole, and eluted with 400 mM imidazole. This purified protein was ft salted using HiTrap Desalting (GE Healthcare).
[0216] (3)プローブ作製 [0216] (3) Probe fabrication
各プロモーターの転写開始点に近い側から 900bpの領域を 30bpづっ区切って、オリ ゴ DNAプローブの配列とした。その際、各プローブとも両隣のプローブ配列と 5bpのォ 一バーラップを持たせて設計したので、 1つのプロモーターあたり合計 40種 (組）のプ ローブペア（センス.アンチセンス）を人工合成した。この際、センス側の DNAを TAMR The region of 900 bp from the side close to the transcription start point of each promoter was divided into 30 bp to obtain the oligo DNA probe sequence. At that time, each probe was designed with both adjacent probe sequences and a 5 bp overlap, so a total of 40 probe pairs (sense / antisense) were artificially synthesized per promoter. At this time, the sense side DNA is
A (蛍光色素)標識した。相互作用の解析には、センス'アンチセンス鎖をァユーリン グさせた 2本鎖 DNAを用いた。アニーリングは、 TAMRA標識センスオリゴ DNA (配列 番号： 173〜332)と未標識アンチセンスオリゴ DNA (配列番号： 333〜492)を、 0.1M NaCl/TE(pH8.0)で luMになるように調整し、 95°Cで 5分変性処理後、 1時間かけて徐 々に室温に戻しながら行った。次に、未反応の 1本鎖オリゴ DNAを除くため、 10mM MgClと Exonuclease Iをカ卩え 37°C、 1時間反応させ MERmaid SPIN Kit (Qbiogene)をA (fluorescent dye) labeled. For the analysis of the interaction, double-stranded DNA in which sense'antisense strands were used was used. For annealing, TAMRA-labeled sense oligo DNA (SEQ ID NO: 173 to 332) and unlabeled antisense oligo DNA (SEQ ID NO: 333 to 492) were adjusted to luM with 0.1M NaCl / TE (pH 8.0). Then, after denaturation treatment at 95 ° C for 5 minutes, it was performed while gradually returning to room temperature over 1 hour. Next, to remove unreacted single-stranded oligo DNA, add 10 mM MgCl and Exonuclease I, react at 37 ° C for 1 hour, and install the MERmaid SPIN Kit (Qbiogene).
2 2
用いてプローブを精製した。センス'アンチセンス鎖の対応は、配列番号： 173の相 補鎖が配列番号： 333であり（Probe No.1)、以降配列番号： 174と 334 (Probe No.2) 〜配列番号： 332と 492 (Probe No.160)のように順番に対応する。  Used to purify the probe. The sense'antisense strand corresponds to the complementary strand of SEQ ID NO: 173 with SEQ ID NO: 333 (Probe No. 1), and thereafter SEQ ID NO: 174 and 334 (Probe No. 2) to SEQ ID NO: 332 Corresponding in order like 492 (Probe No.160).
[0217] (4) DNA-タンパク質結合反応の測定 [0217] (4) Measurement of DNA-protein binding reaction
DNA-タンパク質の結合反応は、 30ulの反応液中 (25mM Hepes/KOH (pH7.9)、 50 mM KC1、 0.5mM DTTゝ 5% glycerol, 2mg/ml BSAゝ 0.05mg/ml poly(dト dC)'(dト dC)、 タンパク質 lul、 3nMプローブ)で室温 20分間行った。分子間相互作用解析機器「一 分子蛍光分析システム MF20 (ォリンパス）」を用いた FCS (Fluorescence Correlation Spectroscopy)計測により、標準 Dyeを 10秒 X 5回、反応液を 15秒 X 5回計測した。この 測定値から並進時間を算出し、結合の有無を検証した。結合が認められる場合は、 標識プローブの分子量が大きくなるため、並進時間がコントロールより長くなる。コント ロールとなる並進時間は、空ベクターを発現させ反応液にそれぞれのプローブを加 えたものの測定値を用いた。 The DNA-protein binding reaction was performed in 30 ul of reaction solution (25 mM Hepes / KOH (pH 7.9), 50 The reaction was performed with mM KC1, 0.5 mM DTT 5% glycerol, 2 mg / ml BSA 0.05 mg / ml poly (d to dC) '(d to dC), protein lul, 3 nM probe) at room temperature for 20 minutes. By using FCS (Fluorescence Correlation Spectroscopy) measurement using an intermolecular interaction analyzer “single molecule fluorescence analysis system MF20 (Olympus)”, standard Dye was measured 10 times X 5 times and reaction solution was measured 15 times X 5 times. The translation time was calculated from this measured value, and the presence or absence of binding was verified. If binding is observed, the molecular weight of the labeled probe will increase, resulting in a longer translation time than the control. For the translation time as a control, the measured value of the empty vector expressed and each probe added to the reaction solution was used.
[0218] (5)木繊維形成遺伝子プロモーターと転写因子との相互作用（結合）  [0218] (5) Interaction (binding) between promoters of wood fiber formation genes and transcription factors
2種の転写因子と 4種のプロモーターの相互作用解析結果を図 5に示す。コント口 ール実験の結果から、少なくとも 20%以上の並進時間の延長がある場合に、相互作 用（結合）があると結論できる。解析結果より、 MYBでは CesAプロモーターの 2ケ所（ 配列番号： 181と 341の 2本鎖 DNA、 199と 359の 2本鎖 DNA)、 CADプロモーターの 1ケ所（配列番号： 246と 406)、 OMTプロモーターの 1ケ所（配列番号： 274と 434)、 C4Hプロモーターの 2ケ所（配列番号： 296と 456、 326と 486)に結合することが示さ れた。 HD- ZIPでは、 CesAプロモーターの 8ケ所（配列番号： 176と 336、 184と 344、 187と 347、 189と 349、 191と 351、 193と 353、 194と 354、 196と 356)、 CADプロ モーターの 4ケ所（酉己歹 IJ番号： 216と 376、 217と 377、 220と 380、 223と 383)、 OM Tプロモーターの 5ケ所（酉己歹 IJ番号： 258と 418、 264と 424、 275と 435、 276と 436、 281と 441)、 C4Hプロモーターの 7ケ所（酉己歹 IJ番号： 301と 461、 307と 467、 313と 4 73、 315と 475、 318と 478、 319と 479、 329と 489)に結合すること力示された。 以上の結果から、本発明の木繊維形成に関わる転写因子群は、木繊維細胞壁形 成に関わる遺伝子群の発現を制御していることが示された。  Fig. 5 shows the results of interaction analysis between the two transcription factors and the four promoters. From the results of the control experiment, it can be concluded that there is an interaction (bonding) when there is a translation time extension of at least 20%. From the results of analysis, in MYB, two sites of CesA promoter (SEQ ID NO: 181 and 341 double-stranded DNA, 199 and 359 double-stranded DNA), one CAD promoter (SEQ ID NO: 246 and 406), OMT promoter 1 (SEQ ID NOs: 274 and 434) and 2 sites of the C4H promoter (SEQ ID NOs: 296 and 456, 326 and 486). In HD-ZIP, 8 locations of CesA promoter (SEQ ID NO: 176 and 336, 184 and 344, 187 and 347, 189 and 349, 191 and 351, 193 and 353, 194 and 354, 196 and 356), CAD promoter 4 locations (IJ numbers: 216 and 376, 217 and 377, 220 and 380, 223 and 383), 5 locations of OM T promoter (IJ numbers: 258 and 418, 264 and 424, and 275 435, 276 and 436, 281 and 441), 7 locations of C4H promoter (IJ: 301 and 461, 307 and 467, 313 and 4 73, 315 and 475, 318 and 478, 319 and 479, 329 and 489) was shown to be capable of binding. From the above results, it was shown that the transcription factor group involved in the formation of the wood fiber of the present invention controls the expression of the gene group involved in the formation of the wood fiber cell wall.
[0219] (実施例 10)転写因子を用いた材質の改変  [0219] (Example 10) Modification of material using transcription factor
木繊維細胞壁形成遺伝子群の発現を制御する配列番号： 101〜111 (配列番号： 112〜122)に記載の転写因子による材質改変効果を確認するため、形質転換タパ コの作出を行った。タバコは草本に分類されるが、基本的な組織構造は榭木と非常 に似ており、榭木の遺伝子解析や導入効果の検証などに一般的に用いられている ( Gray— Mitsumune et al, Plant Mol. Biol. 1999, 39:657—669、 Patzlaff et al, 2003, Pla nt Journal 36: 743-754) 0本実施例では、例として配列番号： 106の MYB転写因子 遺伝子と配列番号： 108のホメォドメイン (HD-ZIP)転写因子遺伝子について、形質転 換体の材質改変結果を示す。 In order to confirm the material modification effect by the transcription factor described in SEQ ID NO: 101 to 111 (SEQ ID NO: 112 to 122) that controls the expression of the tree fiber cell wall-forming gene group, a transformed tapaco was produced. Tobacco is classified as herbaceous, but its basic organizational structure is very similar to that of Kashiwagi, and is generally used for genetic analysis of Kashiwagi and verification of introduction effects ( Gray—Mitsumune et al, Plant Mol. Biol. 1999, 39: 657—669, Patzlaff et al, 2003, Print Journal 36: 743-754) 0 In this example, the MYB transcription factor of SEQ ID NO: 106 is used as an example. The results of modification of the transformant material for the gene and the homeodomain (HD-ZIP) transcription factor gene of SEQ ID NO: 108 are shown.
[0220] (1)材質改変形質転換タバコの作出 [0220] (1) Production of material-modified transgenic tobacco
導入用コンストラクト構築のため、配列番号： 169〜172に記載のプライマーにて、 それぞれの cDNA (配列番号： 106、 108)を铸型に PCRした。増幅された断片の末 端をリン酸化 ·平滑末端化し、予め制限酵素にて GUS遺伝子部分を除き平滑末端ィ匕 した PBI121ベクター (Bevan, M., Nucleic Acids Res. 12, 8711-8721, 1984)の、 35Sプ 口モーター下流にライゲーシヨンし、導入用コンストラクトとした。これらのコンストラクト を、ァグロバタテリゥム（LBA4404株）へエレクト口ポレーシヨンにより導入し、 LB寒天培 地 (カナマイシン、ノ、イダロマイシン各 25 g/ml、 1.0%Agar)で形質転換体を選抜し た。形質転換したァグロバタテリゥムのプレートからシングルコロニーを取り、それぞれ 抗生物質 (カナマイシン、ノ、イダロマイシン各 g/m)を含む LB液体培地中で一 晚培養した（28.5°C, 240rpm) o培養後、ァグロバタテリゥム培養液を遠心（5°C 8000r pm 5min)して集菌し、上清を除き MS液体培地を加え、穏やかに沈殿を懸濁した。懸 濁液を 50mlチューブに集め、懸濁液の OD550が 0.5となるように MS液体培地で希釈 した。この懸濁液を感染操作に用いた。 In order to construct the construct for introduction, each of the cDNAs (SEQ ID NOs: 106 and 108) was subjected to PCR using the primers described in SEQ ID NOs: 169 to 172. PBI121 vector (Bevan, M., Nucleic Acids Res. 12, 8711-8721, 1984) where the end of the amplified fragment was phosphorylated and blunt ended, and the GUS gene portion was removed beforehand using restriction enzymes. This was ligated downstream of the 35S plug motor and used as an introduction construct. These constructs were introduced into Agrobataterum (LBA4404 strain) by electoporation, and transformants were selected using LB agar medium (25 g / ml each of kanamycin, no, and idaromomycin, 1.0% Agar). . Single colonies were picked from the transformed agrobataterum plates and cultured in LB liquid medium containing antibiotics (kanamycin, no, idaromomycin each g / m) (28.5 ° C, 240 rpm) o culture Thereafter, the agrobacterium was collected by centrifugation (5 ° C., 8000 rpm 5 min), the supernatant was removed, and MS liquid medium was added to gently suspend the precipitate. The suspension was collected in a 50 ml tube and diluted with MS liquid medium so that the OD550 of the suspension was 0.5. This suspension was used for the infection procedure.
[0221] 感染用の試料としては、生育用培地（MS 3%Sucrose N7B5* l%Agar pH5.6)にて無 菌で育てたタバコの若い葉を用いた。滅菌水を入れたシャーレ内に葉を置き、メスで 葉の周囲と主葉脈を除き、残りの部分を約 7mm角に切った。それらの切片を滅菌キム タオルにはさんで軽く脱水し、葉の裏面を上にして前培養培地（MS, 3%Sucrose, NA A 10— 7mM, BA = 10— 5mM, l%Agar, pH5.6 )に並べ、 25°Cにてー晚前培養した。 [0221] As a sample for infection, young leaves of tobacco grown in a sterilized culture medium (MS 3% Sucrose N7B5 * 1% Agar pH 5.6) were used. Leaves were placed in a petri dish containing sterilized water, the circumference of the leaves and the main veins were removed with a scalpel, and the remaining part was cut into approximately 7 mm squares. Lightly dehydrated across those sections in sterile Kim towels, pre-culture medium in the top rear surface of the leaf (MS, 3% Sucrose, NA A 10- 7 mM, BA = 10- 5 mM, l% Agar, The cells were aligned at pH 5.6) and pre-cultured at 25 ° C.
[0222] 前培養した葉の切片をァグロの懸濁液に浸し、 20分間軽く振とう培養した後、切片 を滅菌キムタオルではさんで軽く水分を除き、葉の裏面を上にして共存培地に並べ、 暗黒下 25°Cで 2〜3日共存培養した。共存培養した切片をピンセットで選択培地に移 し、明所 25°Cで培養した。培養開始以降、 2週間ごとに新しい選択培地に移植し、シ ユートが再分化してきたらホルモンフリーの発根培地（1/2MS 1.5%Sucrose l%Agar pH 5.6 CU200 g/ml Hyg30 g/ml)に移植し、形質転換体の選抜を行った。 [0222] After pre-cultured leaf sections were soaked in agro suspension and gently shaken for 20 minutes, the sections were sandwiched between sterile Kim towels to remove moisture, and placed in the co-culture medium with the back of the leaves facing up. The cells were co-cultured at 25 ° C for 2 to 3 days in the dark. The co-cultured sections were transferred to the selective medium with tweezers and cultured at 25 ° C in the light. After the start of culture, transplant to a new selective medium every two weeks, and when the stem has re-differentiated, hormone-free rooting medium (1 / 2MS 1.5% Sucrose l% Agar pH 5.6 CU200 g / ml Hyg30 g / ml), and transformants were selected.
[0223] (2)材質改変形質転換タバコの解析 [0223] (2) Analysis of material-modified transgenic tobacco
配列番号： 106の MYB転写因子遺伝子と配列番号： 108のホメォドメイン (HD- ZIP) 転写因子遺伝子を過剰発現した形質転換タバコ植物体を図 6に示す。形質転換体 の材質分析として、リグニン含量と繊維長の測定を行った。リグニンの定量 (酸可溶性 '不溶性リグニンの総量）は Klason法（木材化学実験書 II.化学編 P150-155、 Patzlaff et al., 2003, Plant Journal)で、繊維長の測定は Jefrey法（Han et al., 1999, Kenaf Pr operties, Processing and Products. P149- 167)で行った。その結果、 MYB転写因子 導入個体では、リグニンの絶乾重量あたりの割合が平均 14.7± 1.8% (5個体平均)で コントロール（野生株）の 19.3± 1.1%と比べ減少していた。また、繊維長はコントロール の 560.6±50.9umであるのに対し、 MYB導入個体では 620.3 ±42. lumと長くなつてい た。絶乾重量に対するリグニン割合の低下は、相対的にセルロース割合の増加を示 す。従って、 MYB転写因子を過剰発現することにより、セルロースが多く長い繊維長 の材質に改変することができた。このことは、 RNAi等の技術を用いて、本転写因子の 発現を抑制すれば、逆の材質 (セルロースが少なく短い繊維長）に改変できることも 示された。  FIG. 6 shows a transformed tobacco plant overexpressing the MYB transcription factor gene of SEQ ID NO: 106 and the homeodomain (HD-ZIP) transcription factor gene of SEQ ID NO: 108. As a material analysis of the transformant, lignin content and fiber length were measured. Lignin quantification (total amount of acid-soluble 'insoluble lignin) was determined by the Klason method (Wood Chemistry Experiments II. Chemistry P150-155, Patzlaff et al., 2003, Plant Journal). al., 1999, Kenaf Pro operties, Processing and Products. P149-167). As a result, in the MYB transcription factor-introduced individuals, the ratio of lignin per bone dry weight averaged 14.7 ± 1.8% (average of 5 individuals) compared to 19.3 ± 1.1% of the control (wild strain). The fiber length was 560.6 ± 50.9um for the control, whereas it was 620.3 ± 42.lum for the MYB-introduced individuals. A decrease in the lignin ratio relative to the absolute dry weight indicates a relative increase in the cellulose ratio. Therefore, by overexpressing the MYB transcription factor, it was possible to modify the material with a long fiber length with a large amount of cellulose. This indicates that if the expression of this transcription factor is suppressed using a technique such as RNAi, the material can be altered to the opposite material (short fiber length with less cellulose).
[0224] 一方、 HD-ZIP転写因子導入個体では、リグニンの割合が平均 23.1 ±2.5% (5個体 平均）で増加していた力 繊維長は 534.6±34.3umで短くなつていた。このように、 HD -ZIP転写因子を過剰発現することにより、セルロースが少なく短い繊維長の材質に改 変できた。同時に、本転写因子の発現を抑制すれば、逆の材質 (セルロースが多く長 い繊維長）に改変できることも示された。  [0224] On the other hand, in the HD-ZIP transcription factor-introduced individuals, the lignin ratio increased at an average of 23.1 ± 2.5% (average of 5 individuals), and the fiber length decreased at 534.6 ± 34.3um. Thus, by overexpressing the HD-ZIP transcription factor, it was possible to change to a material with less cellulose and a shorter fiber length. At the same time, it was shown that if the expression of this transcription factor is suppressed, it can be changed to the opposite material (long fiber length with much cellulose).
以上の結果から、本願で示された転写因子による植物の材質改変効果が示された  From the above results, plant material modification effect by the transcription factor shown in this application was shown.
[0225] (実施例 11)木繊維細胞壁形成遺伝子群を用いた植物榭幹木部の生長状態と種の 検査 [Example 11] Examination of growth state and species of plant stems using tree fiber cell wall forming genes
(1)ユーカリ榭幹の地上高による材質の変動 (垂直変動）  (1) Material variation due to ground clearance of Eucalyptus trunk (vertical variation)
木繊維性質の主要要素である細胞壁成分 ·導管分布密度 ·繊維長等については、 広葉樹 '針葉樹を問わず、地上高により変動する組織形成を行うことが知られている 。この地上高による木繊維性質の変動現象については、古くから榭幹内の材質の垂 直変動として知られる。各地上高 (基部 ·中央部，榭冠部)を相互比較すると、中央部 (胸高部以上〜榭冠部未満)では細胞壁成分については、セルロースが増大し、リグ ニン、へミセルロースが減少する。また、形態的観察では、導管分布密度が少なぐ 繊維長は約 20%の増加が認められる。さらに、細胞壁の厚さは、榭冠部から基部に 向かって増加していく。詳細は不明であるが、各地上高の木繊維形成組織の成熟度 合いにより、形成される木繊維性質の差が発生しているものと考えられている（島地 謙 ·須藤彰司，原田浩著「木材の組織」、古野毅 ·澤辺攻著「木材科学講座 2 ·組織と 材質」）。 Cell wall components, conduit distribution density, fiber length, etc., which are the main elements of wood fiber properties, are known as broad-leaved trees. . This fluctuation phenomenon of wood fiber properties due to ground clearance has long been known as vertical fluctuation of the material in the trunk. Comparing each ground height (base · middle, crown), cellulose increases in cell wall components, and lignin and hemicellulose decrease in the center (above the chest and less than crown). . In addition, morphological observation shows that the fiber length with a small conduit distribution density increases by about 20%. Furthermore, the cell wall thickness increases from the crown to the base. Although details are unknown, it is considered that the difference in the properties of the wood fiber formed depends on the maturity of the wood fiber-forming structures above ground (Ken Shimaji, Akiji Sudo, Hiroshi Harada) Written by “Wood Organization”, Satoshi Furuno · Satoshi Sawabe “Wood Science Course 2 • Organization and Materials”).
[0226] 本発明者らは、一般的に言われている材質の垂直変動を確認するため、ユーカリ 属カマルドレンシス種の 5年生クローン系統（CPT1)を材料に、以下に示す実施例の ように材質分析を行った。一般的に、 5年生のカマルドレンシスは、榭幹基部では低 生長で未熟な木繊維 (未熟材:短繊維、直径は細ぐ細胞壁は薄ぐフィブリル傾角 は急で、低木繊維率と低比重)を形成すること、中央部では高生長で成熟した木繊 維 (成熟材:長繊維、直径は太ぐ細胞壁は厚ぐフィブリル傾角は緩ぐ高木繊維率 と高比重）を形成することが知られている。この特徴は、伐採される 5〜： LO年生の力マ ルドレンシスに共通している。  [0226] In order to confirm the vertical variation of the generally-known material, the present inventors used a 5-year-old clone line (CPT1) of the Eucalyptus genus Camaldrensis as a material as in the following examples. Material analysis was conducted. In general, 5-year-old chamaldrensis is low growth and immature wood fiber at the trunk base (immature wood: short fiber, diameter is thin, cell wall is thin, fibril inclination is steep, shrub fiber rate and low specific gravity In the middle, it is known that a high-growth and mature wood fiber (mature wood: long fibers, thick cell walls are thick, fibril inclination is loose, and wood fiber ratio and high specific gravity) is formed. It has been. This characteristic is common to power harvesting of 5th grade LO:
[0227] 5年生の植物体の基部（地上高 lm未満）と中央部（地上高約 5m)の榭幹を用いた 。それぞれの榭幹カも榭皮を除き、厚さ 25mmのディスクに切り分け十分に乾燥させ た後、粉砕してチップにしチップミキサーで 3分間よく混合し分析に用いた。パルプ精 製と材質分析の方法は、基本的に紙パルプ技術協会発行 [紙パルプ製造技術シリ ーズ]第 1卷「クラフトパルプ」、第 3卷「パルプの洗浄 '精選'漂白」、第 9卷「紙パルプ の試験法」に記載されている常法に従った。蒸解は、 13.2%の Na Sにて 170°Cで 90分  [0227] The trunks of the base (less than lm above the ground) and the central part (about 5 m above the ground) of the fifth grade plant were used. Each carcass was also cut into 25mm-thick discs after removing the crust and dried, then crushed into chips and mixed well with a chip mixer for 3 minutes for analysis. The pulp refining and material analysis methods are basically published by the Paper Pulp Technology Association [Paper Pulp Manufacturing Technology Series] 1st “Kraft Pulp”, 3rd “Pulp Washing 'Selection' Bleaching”, 9th常 The usual method described in “Paper pulp test method” was followed. Cooking is 90 minutes at 170 ° C with 13.2% Na S
2  2
間行 、、カッパ一価 18を基準としてパルプデータを補正した。  In the meantime, the pulp data was corrected based on the kappa price of 18.
[0228] 前述の方法によって得られた材質分析の結果、 5年生ユーカリ植物体の基部では、 容積重が 432kg/m2、パルプ収率が 43.8%、繊維長が 0.66mmであった。一方、中央 部では容積重が 525kg/m2、パルプ収率が 53.4%、繊維長が 0.86mmであった。これら の結果から、一般的に言われている垂直変動力 本実施例のサンプルでも確認され た。また、材質としては高容積重'高パルプ収率で長い繊維を持つ中央部の方が優 れて 、ることも確認された。 [0228] As a result of the material analysis obtained by the above-mentioned method, the bulk weight of the 5th-year eucalyptus plant was 432 kg / m 2 , the pulp yield was 43.8%, and the fiber length was 0.66 mm. On the other hand, in the central part, the bulk weight was 525 kg / m 2 , the pulp yield was 53.4%, and the fiber length was 0.86 mm. From these results, it is confirmed in the sample of this example that the vertical fluctuation force is generally called. It was. In addition, it was confirmed that the center part having high fiber weight and high pulp yield and long fibers was superior as the material.
[0229] (2)地上高による発現プロファイル差に基づいた材質の検査 (発現プロファイルの比 較）  [0229] (2) Inspection of materials based on difference in expression profile due to ground clearance (comparison of expression profiles)
実施例 3によって示された 34遺伝子と実施例 6によって示された 11遺伝子の、地 上高の違いによる発現プロフィール分類表に従 、、被検サンプルの材質検査を行つ た。被検サンプルとしては、 2個体（Sl、 2)の 7年生ユーカリカマルドレンシスの榭幹 中央部を用いた。 RNAの抽出、マイクロアレイ実験は実施例 1〜3に、材質分析は前 述の方法に従った。得られた各サンプルの発現プロフィールを表 2の分類群と比較し 、基部側のプロフィールに似ているのか中央部側のに似ているのかを調べることで、 被検サンプルの材質が未熟材に近 ヽ (質が劣る）のか、成熟材に近!ヽ (質が優れて いる）のかが判別できる。被検サンプル部位は、榭幹基部でも中央部でもそれ以外の どこでも良い。榭幹中央部を使った場合、その部分が個体内で最も優れた材質を持 つていると一般的に予想されるので、判別された材質はその個体内の最良の材質で あると言える。榭幹基部の場合は、その逆である。  According to the expression profile classification table according to the difference in ground height between the 34 genes shown in Example 3 and the 11 genes shown in Example 6, the materials of the test samples were examined. As the test sample, the center of trunk of 2 individuals (Sl, 2) of 7th grade Eucalyptus maldorensis was used. RNA extraction and microarray experiments were performed in Examples 1 to 3, and material analysis was performed according to the method described above. By comparing the expression profile of each obtained sample with the taxon of Table 2, it is possible to make the material of the sample to be immature by examining whether it is similar to the profile on the base side or the center side. It is possible to determine whether it is close (inferior in quality) or close to matured material! The sample site to be examined can be at the trunk base, at the center, or anywhere else. When the trunk center is used, it is generally expected that the part will have the best material in the individual, so it can be said that the identified material is the best material in the individual. The opposite is true for trunk bases.
[0230] (3)サンプル 1 (S1)の検査（木部 vs師部のプロフィール） [0230] (3) Examination of sample 1 (S1) (Kibe vs. phloem profile)
表 2と 6で逆相関、 cam5特異的に分類された 12遺伝子の、 S1での発現ブロフィー ルは以下のようであった。  The expression profiles in S1 of 12 genes classified inversely in Tables 2 and 6 and cam5 specific were as follows.
逆相関を示す 1遺伝子のプロフィール：基部的なプロフィール（X〉P)を示した。 cam5 (中央部）特異的に X〉Pを示す 11遺伝子：基部的なプロフィール (X=P)の遺伝 子 9個、中央部的なプロフィール（X〉P)の遺伝子は 2個であった。  A single gene profile showing an inverse correlation: a basic profile (X> P) was shown. Eleven genes that specifically show cam> (central part): X> P: Nine genes with basic profile (X = P), and two genes with central profile (X> P).
[0231] 総合すると、基部的なプロフィールの遺伝子の総数は全体の 83.3%にあたる 11個で 、中央部的なプロフィールの遺伝子の総数は、 16.7%にあたる 2個であった。以上の 結果から、 S1のプロフィールは基部に非常に近いがやや中央部的であることがわか つた。したがって、 S1の材質は caml (基部）よりも少し優れていると判別される。 [0231] Overall, the total number of genes in the basic profile was 11 or 83.3%, and the total number of genes in the central profile was 2 or 16.7%. From the above results, it was found that the profile of S1 is very close to the base but somewhat central. Therefore, the material of S1 is determined to be a little better than caml (base).
[0232] (4)サンプル 2 (S2)の検査（木部 vs師部のプロフィール） [0232] (4) Examination of sample 2 (S2) (Kibu vs. Shimobu profile)
表 2と 6で逆相関、 cam5特異的に分類された 12遺伝子の、 S2での発現ブロフィー ルは以下のようであった。 逆相関を示す 1遺伝子のプロフィール：基部的なプロフィール（X〉P)を示した。 cam5 (中央部）特異的に X〉Pを示す 11遺伝子：基部的なプロフィール (X=P)の遺伝 子 6個、中央部的なプロフィール（X〉P)の遺伝子は 5個であった。 The expression profiles in S2 of 12 genes that were inversely correlated in Tables 2 and 6 and classified cam5 specific were as follows. A single gene profile showing an inverse correlation: a basic profile (X> P) was shown. 11 genes specifically showing cam> (center) X> P: 6 genes with basic profile (X = P) and 5 genes with central profile (X> P).
[0233] 総合すると、基部的なプロフィールの遺伝子の総数は、全体の 58.3%にあたる 7個で 、中央部的なプロフィールの遺伝子の総数は、 41.7%のあたる 5個であった。この結果 から、 S2のプロフィールは基部的ではあるがかなり中央部的な要素が含まれているこ とがわかった。したがって、 S2の材質は caml (基部）と cam5 (中央部）のほぼ中間程 度と判別される。 [0233] Overall, the total number of genes in the basic profile was 7 (58.3%), and the total number of genes in the central profile was 5 (41.7%). From this result, it was found that the profile of S2 contains a basic but fairly central element. Therefore, the material of S2 is determined to be approximately halfway between caml (base) and cam5 (center).
[0234] (5)サンプル 1と 2の相対比較（木部 vs木部のプロフィール）  [0234] (5) Relative comparison of samples 1 and 2 (Kibe vs. Kibe profile)
S 1と S 2の材質を相対比較するため、 S 1と S 2の木部間でマイクロアレイ実験を行つ た。その結果、表 2と 6で木部間で差があった 22遺伝子 (2+18+2)の発現プロフィール は、以下のようであった。  In order to make a relative comparison between the materials of S1 and S2, a microarray experiment was performed between the xylems of S1 and S2. As a result, the expression profiles of 22 genes (2 + 18 + 2) that differed between xylem in Tables 2 and 6 were as follows.
X〉Pでかつ 1〉5である 2遺伝子のプロフィール： caml (材質が悪い方）で発現が強い 本グループの遺伝子は、 2遺伝子とも S1〉S2で S1の方が強発現であった。  Profiles of 2 genes with X> P and 1> 5: Strong expression in caml (poor material) Both genes in this group were S1> S2 and S1 was more strongly expressed.
X〉Pでかつ 1〈5である 20遺伝子： cam5 (材質が良 、方）で発現が強 、本グループ では、 S1〉S2の遺伝子が 1個、 S1=S2の遺伝子は 5個、 S1〈S2の遺伝子は 14個で、 S2 で多く発現していた。  20 genes with X> P and 1 <5: cam5 (material is good, one) has strong expression. In this group, there is 1 gene with S1> S2, 5 genes with S1 = S2, S1 < There were 14 S2 genes, and many were expressed in S2.
[0235] 全体として caml (材質が悪 、方)で発現が強 、遺伝子群は S1で、 cam5 (材質が良 V、方)で発現が強 、遺伝子群は S2で多く発現して 、たことから、 S2の方が S 1よりも 優れた材質を持っていると判別される。（3) (4)の検査では、 S1に比べより中央部に 近 、と判断された S2が材質的に優れて 、ると 、う結果であった。木部同士の直接比 較の結果も、それを支持する結果であった。  [0235] As a whole, the expression was strong in caml (bad material, one), the gene group was S1, the expression was strong in cam5 (good material, V), and the gene group was highly expressed in S2. From this, it is determined that S2 has a better material than S1. (3) In the inspection of (4), S2 was judged to be closer to the center than S1, and the result was that S2 was superior in material quality. The result of direct comparison between the xylem was also the result that supported it.
[0236] (6)被検サンプルの材質分析  [0236] (6) Material analysis of test sample
S1と S2の材質を、実施例 11 (1)に記載の方法で分析した。その結果、容積重が S 1で 449kg/m2、 S2で 488kg/m2、パルプ収率が SIで 45.7%、 S2で 50.1%、繊維長が S1で 0.69mm、 S2で 0.75mmであった。したがって、材質は前述の検査結果に基づく 予想通り、 S1は基部に近い材質であり、 S2は基部的ではあるがやや中央部的な材 質であった。また、両方を比較すると、予想通り S2の方が相対的に優れていた。この ように、本願で示された発現プロフィールに基づく比較により、生きたまま被検サンプ ルの材質を判別することができる。 The materials of S1 and S2 were analyzed by the method described in Example 11 (1). As a result, 488kg / m 2 at 449kg / m 2, S2 in test weight is S 1, 45.7% pulp yield in SI, 50.1% in S2, 0.69 mm fiber length by S1, was 0.75mm in S2 . Therefore, as expected based on the above-mentioned inspection results, S1 was a material close to the base, and S2 was a basic but somewhat central material. Also, comparing both, S2 was relatively better as expected. this As described above, the material of the sample to be tested can be discriminated alive by comparison based on the expression profile shown in the present application.
[0237] (7)ユーカリ榭幹の種による材質の変動 (榭種間変動）  [0237] (7) Variation of material by species of eucalyptus trunk (varietal variation)
木繊維性質の主要要素である細胞壁成分 ·導管分布密度 ·繊維長等については、 広葉樹 '針葉樹を問わず、種により変動する組織形成を行うことが知られている（島地 謙 ·須藤彰司，原田浩著「木材の組織」、古野毅 ·澤辺攻著「木材科学講座 2 ·組織と 材質」）。この種による木繊維性質の変動現象については、古くから材質の榭種間変 動として知られる。  Cell wall components, conduit distribution density, fiber length, etc., which are the main elements of wood fiber properties, are broad-leaved trees. It is known to form a tissue that varies depending on the species, regardless of the conifer (Ken Shimaji, Akiji Sudo, Hiroshi Harada “Wood Organization”, Satoshi Furuno · Satoshi Sawabe “Wood Science Course 2 • Organization and Materials”). This variation in wood fiber properties due to this species has long been known as the variation between species of wood.
[0238] 例えば、ユーカリ属'カマルドレンシス種とグロブルス種を比較すると、一般的に、グ ロブルス種の方力 セルロースが増大し、リグニン、へミセルロースが減少する。また、 形態的観察では、導管分布密度が少なぐ繊維長は増加が認められる。さらに、細胞 壁は厚い。詳細は不明であるが、種によって木繊維形成組織の性質が異なり、形成 される木繊維性質の差が発生して ヽるものと考えられて ヽる。  [0238] For example, comparing Eucalyptus genus' Camardolensis and Globulus species, the cellulose of the Globules species generally increases cellulose, while lignin and hemicellulose decrease. In addition, morphological observation shows an increase in fiber length when the conduit density is low. In addition, the cell wall is thick. The details are unknown, but the nature of the wood fiber-forming tissue differs depending on the species, and it is thought that the difference in the nature of the wood fiber formed is caused.
[0239] 本発明者らは、一般的に言われている材質の榭種間変動を確認するため、 5年生 のユーカリ属カマルドレンシス種クローン系統（CPT1)とグロブルス種クローン系統（G AL1)を材料に、実施例 2に示す方法で材質分析を行った。一般的に、 5年生の力マ ルドレンシスの榭幹では、グロプルスに比べ劣った木繊維 (低品質パルプ：短繊維、 直径は細ぐ細胞壁は薄ぐフィブリル傾角は急で、低木繊維率と低比重）を形成す ること、 5年生のグロプルスの榭幹では、優れたパルプ (長繊維、直径は太ぐ細胞壁 は厚ぐフィブリル傾角は緩ぐ高木繊維率と高比重）を形成することが知られている。 この特徴は、伐採される 5〜10年生のカマルドレンシスおよびグロブルスも同じである  [0239] In order to confirm the variation between the species of the generally-known materials, the present inventors have established a 5-year-old Eucalyptus Camaldorensis Clone Line (CPT1) and a Globulus Clone Line (GAL1). The materials were analyzed by the method shown in Example 2 for the materials. In general, in the trunk of the fifth grade power maldrensis, wood fibers that are inferior to those of Gloples (low-quality pulp: short fibers, thin cell walls are thin, fibril inclination is steep, the percentage of shrub fibers and low specific gravity are low. It is known that 5th grade Groplus trunks form excellent pulp (long fibers, thicker cell walls are thicker and thicker fibril tilt is looser wood and higher specific gravity). ing. This characteristic is the same for 5-10 grade camaldrensis and globules that are harvested.
[0240] 本発明においては、前述の 5年生のユーカリ属カマルドレンシスの榭幹基部とユー カリ属グロブルスの榭幹基部（共に地上高 lm未満）を用い、前述に記載のクラフトパ ルプ精製の定法に従った。材質分析の結果、 5年生カマルドレンシス植物体の基部 では、容積重が 432kg/m2、パルプ収率が 43.8%、繊維長が 0.66mmであった。一方、 グロプルスの榭幹基部では容積重が 574kg/m2、パルプ収率が 57.1%、繊維長が 0.95 mmであった。これらの結果から、一般的に言われている榭種間変動力 本実施例の サンプルでも確認された。また、材質としては高容積重で長い繊維を持つグロプルス 榭幹基部の方が優れて 、ることも確認された。 [0240] In the present invention, the above-mentioned conventional method for refining kraft pulp using the trunk base of Eucalyptus genus Camaldrensis and the trunk base of Eucalyptus globulus (both above ground level lm) are used. Followed. As a result of material analysis, at the base of the 5-year-old camaldrensis plant, the bulk weight was 432 kg / m 2 , the pulp yield was 43.8%, and the fiber length was 0.66 mm. On the other hand, the gross base of Groplus had a bulk weight of 574 kg / m 2 , a pulp yield of 57.1%, and a fiber length of 0.95 mm. From these results, it is generally said that the variability between species varies. It was also confirmed in the sample. In addition, it was also confirmed that the GLOPLUS trunk base with high bulk weight and long fibers was superior as the material.
[0241] (8)種間での発現プロファイル差に基づ 、た材質の検査 (発現プロファイルの比較） 実施例 3によって示された 34遺伝子と実施例 6によって示された 11遺伝子の、種の 違いによる発現プロフィール分類表に従い、被検サンプルの材質検査を行った。被 検サンプルとしては、 7年生ユーカリカマルドレンシス（C1 :実施例 11 (3)の S1と同じ )と 7年生ユーカリグロプルス（G1)を各 1個体用いた。 RNAの抽出、マイクロアレイ実 験は実施例 1〜3に、材質分析は実施例 11 (1)の方法に従った。得られた各サンプ ルの発現プロフィールを表 3と 7の分類群と比較し、カマルドレンシスのプロフィール に似ているのか、グロプルスのそれに似ているのかを調べることで、被検サンプルの 種と材質 (カマルドレンシス的かグロプルス的カゝ）が判別できる。被検サンプル部位は 、榭幹基部でも中央部でもそれ以外のどこでも良い。  [0241] (8) Examination of material based on expression profile difference between species (Comparison of expression profiles) of 34 genes shown in Example 3 and 11 genes shown in Example 6 According to the expression profile classification table according to the difference, the material test of the test sample was performed. As test samples, 7 individual eucalyptus maldrensis (C1: same as S1 in Example 11 (3)) and 7 year eucalyptus globulus (G1) were used. RNA extraction and microarray experiments were carried out in accordance with Examples 1 to 3, and material analysis was carried out in accordance with the method of Example 11 (1). By comparing the expression profile of each sample obtained with the taxa in Tables 3 and 7, and examining whether it is similar to the profile of chamaldrensis or that of globus, The material (camaldrensis or glopuls) can be distinguished. The sample site to be examined may be the trunk base, the center, or any other location.
[0242] (9)カマルドレンシスサンプル (C1)の検査（木部 vs師部のプロフィール） [0242] (9) Examination of the Camaldorensis sample (C1) (Kibe vs. phloem profile)
表 3と 7で逆相関、 cam特異的に分類された 23遺伝子の C1での発現プロフィール は以下のようであった。  The expression profiles in C1 of 23 genes that were inversely correlated and cam-specifically classified in Tables 3 and 7 were as follows.
逆相関を示す 9遺伝子のプロフィール：カマルドレンシス的なプロフィール（X〉P)を 示す遺伝子は 8個、グロプルス的なプロフィール（Xく P)を示す遺伝子は 1個であった カマルドレンシス特異的に X〉Pを示す 14遺伝子：カマルドレンシス的（X〉P)な遺伝 子は 12個、グロブノレス的（X=P)な遺伝子は 2個であった。  Profile of 9 genes showing inverse correlation: 8 genes with camaldrense-like profile (X> P) and 1 gene with globulous-like profile (X P P) Camaldrensis-specific There are 14 genes that show X> P: 12 genes that are chamaldrensic (X> P) and 2 genes that are Globnores (X = P).
[0243] 総合すると、カマルドレンシス的なプロフィールの遺伝子の総数は、全体の約 87.0% にあたる 20個で、グロプルス的なプロフィールの遺伝子の総数は、 13.0%のあたる 3個 であった。この結果から、 C1のプロフィールはカマルドレンシス的ではある力 若干グ ロブルス的な要素も多く含むことがわかった。したがって、 C1の種は確かにカマルド レンシスであるが、材質は一般的なカマルドレンシスよりもややグロブルス的（良質） であると判別される。  [0243] Taken together, the total number of genes with a camaldrenetic profile was 20 or about 87.0% of the total, and the total number of genes with a Glopus profile was 3 or 13.0%. From this result, it was found that the profile of C1 contains a lot of forces that are camaldrensis and some globules. Therefore, although the C1 seed is certainly chamaldrensis, it is determined that the material is a little more globules (good quality) than general chamaldrensis.
[0244] (10)グロプルスサンプル (G1)の検査（木部 vs師部のプロフィール）  [0244] (10) Examination of the Gropulus sample (G1) (Kibu vs. phloem profile)
表 3と 7で逆相関、 cam特異的に分類された 23遺伝子の G1での発現プロフィール は以下のようであった。 Inverse correlation in Tables 3 and 7, G1 expression profiles of 23 genes classified cam specific Was as follows.
逆相関を示す 9遺伝子のプロフィール：カマルドレンシス的なプロフィール（X〉P)を 示す遺伝子は 1個、グロプルス的なプロフィール（Xく P)を示す遺伝子は 8個であった カマルドレンシス特異的に X〉Pを示す 14遺伝子：カマルドレンシス的（X〉P)な遺伝 子は 4個、グロブルス的（X=P)な遺伝子は 10個であった。  Nine gene profiles showing inverse correlation: one gene with a camaldrenetic profile (X> P) and eight genes with a globulous profile (X P P) There are 14 genes with X> P: 4 genes with chamaldrensis (X> P) and 10 genes with Globulus (X = P).
[0245] 総合すると、カマルドレンシス的なプロフィールの遺伝子の総数は、全体の 21.7%に あたる 5個で、グロプルス的なプロフィールの遺伝子の総数は、 78.3%のあたる 18個で あった。この結果から、 G1のプロフィールはグロブルス的であることがわかった。した がって、 G1の種は確かにグロプルスである力 材質は一般的なグロプルスよりも力マ ルドレンシス的 (低質)であると判別される。 [0245] Taken together, the total number of genes with a camaldrenetic profile was 5 or 21.7%, and the total number of genes with a globus profile was 18 or 78.3%. From this result, it was found that the profile of G1 is Globulous. Therefore, it is determined that the force material whose G1 species is certainly globules is more force-mallensic (low quality) than general glops.
[0246] (11)じ1と01の相対比較(木部 vs木部のプロフィール） [0246] (11) Relative comparison of 1 and 01 (Kibe vs. Kibe profile)
C 1と G 1の木部のプロフィールを直接比較するため、 C 1と G 1の木部間でマイクロ アレイ実験を行った。その結果、表 3と 7で木部間で差があった 6遺伝子 (4+1+1)の発 現プロフィールは、以下のようであった。  To directly compare the xylem profiles of C 1 and G 1, microarray experiments were performed between C 1 and G 1 xylem. As a result, the expression profiles of 6 genes (4 + 1 + 1) that differed between xylem in Tables 3 and 7 were as follows.
X〉Pでかつ cam〉gloである 4遺伝子： C1〉G1の遺伝子は 5個、 C1=G1の遺伝子は 1個 、 C1〈G1の遺伝子は 2個で、カマルドレンシスで発現が強い本グループの遺伝子は、 C1で多く発現していた。  4 genes that are X> P and cam> glo: 5 C1> G1 genes, 1 C1 = G1 gene, 2 C1 <G1 genes, and this group is highly expressed by camaldrensis The gene was highly expressed in C1.
X〉Pでかつ camく gloである 2遺伝子：グロプルスで発現が強!、本グループの遺伝子 は、 2遺伝子とも C1く G1の遺伝子で、 G1で多く発現していた。  Two genes: X> P and cam glo: Strongly expressed in globus! Both genes in this group are C1 and G1, and many genes were expressed in G1.
[0247] 全体としてカマルドレンシスで発現が強い遺伝子群は C1で、グロプルスで発現が 強い遺伝子群は G1で多く発現していたことから、木部同士の直接比較の結果も、 (9 ) (10)の検査と同様に、 C1はカマルドレンシスで、 G1がグロプルスであると判別され た。したがって、材質もグロブルスである G1が優れていると推定できる。 [0247] As a whole, the gene group that is strongly expressed by camaldrensis was expressed in C1, and the gene group that was strongly expressed in Gloples was expressed in G1. Similar to the test in 10), C1 was determined to be camaldrensis and G1 was determined to be globus. Therefore, it can be presumed that the material G1 is also excellent.
[0248] (12)材質の分析 [0248] (12) Material analysis
C1と G1の材質を、実施例 11 (1)に記載の方法で分析した。その結果、容積重が C 1で 449kg/m2、 G1で 534kg/m2、パルプ収率が C1で 45.7%、 G1で 53.6%、繊維長が C1で 0.69mm、 G1で 0.86mmであった。したがって、材質は前述の検査結果に基づく 予想通り、 CIがカマルドレンシス的な材質であり、 G1はグロプルス的な材質であった 。また、両方を比較すると、予想通り G1の方が相対的に優れていた。このように、本 願で示された発現プロフィールに基づく比較により、生きたまま被検サンプルの種と 材質を判別することができる。 The materials of C1 and G1 were analyzed by the method described in Example 11 (1). As a result, 534kg / m 2 at 449kg / m 2, G1 in test weight is C 1, 45.7% for C1 pulp yield 53.6% for G1, fiber length was 0.86mm in C1 0.69 mm, in G1 . Therefore, the material is based on the above inspection results As expected, CI was a camaldrensis-like material and G1 was a globus-like material. Also, comparing both, G1 was relatively better as expected. In this way, the species and material of the test sample can be discriminated alive by comparison based on the expression profile shown in the present application.
[0249] 本発明者らは、作製したユーカリ ESTデータベースを用いて、マイクロアレイ解析に よるユーカリ木繊維形成組織において細胞壁合成に関与する遺伝子群の抽出と、そ れらの地上高 ·種の違いによる発現変動を調べた。その結果、ユーカリ木繊維形成 組織で発現して!/ヽる細胞壁合成遺伝子群を特定し、地上高 ·種の違!ヽで優位に発現 が変動する遺伝子群を同定した。さらに、それらのプロモーター DNAを取得し、木部 発現用プロモーターとして機能することを確認した。上記遺伝子群と発現情報'プロ モーター DNAは、木繊維形成組織の細胞壁合成および形態形成の制御に利用でき る。 [0249] Using the prepared eucalyptus EST database, the present inventors extracted a group of genes involved in cell wall synthesis in a eucalyptus tree fiber-forming tissue by microarray analysis, and the difference in the height and species of the ground. Expression variation was examined. As a result, we identified a cell wall synthetic gene group that is expressed / excited in eucalyptus tree fiber-forming tissues, and a gene group whose expression fluctuates predominantly due to differences in the height and species of the ground. Furthermore, these promoter DNAs were obtained and confirmed to function as promoters for xylem expression. The above gene group and expression information 'promoter DNA can be used to control cell wall synthesis and morphogenesis in wood fiber-forming tissues.
[0250] また、本発明者らは、作製したユーカリ ESTデータベースを用いて、マイクロアレイ 解析による木繊維形成に関わる転写因子群の抽出と、それらの地上高 ·種の違いに よる発現変動を調べた。その結果、ユーカリ木繊維形成に関わる転写因子群を特定 し、地上高 ·種の違いで優位に発現が変動する遺伝子群を同定しそれらのプロモー ター DNAを取得した。さらに、それらの転写因子を導入することで、植物の材質の改 変できることを示した。上記遺伝子セットと発現情報'プロモーター DNAは、木繊維形 成 (材質)の制御に利用できる。  [0250] In addition, the present inventors examined the expression variation due to the difference in the height and species of the ground using the prepared eucalyptus EST database to extract a group of transcription factors involved in tree fiber formation by microarray analysis. . As a result, we identified a group of transcription factors involved in eucalyptus tree fiber formation, identified a group of genes whose expression fluctuated predominantly depending on the height of the ground, and obtained their promoter DNA. Furthermore, we showed that plant materials can be altered by introducing these transcription factors. The above gene set and expression information 'promoter DNA can be used to control wood fiber formation (material).
図面の簡単な説明  Brief Description of Drawings
[0251] [図 1]ユーカリ属カマルドレンシス種の 5年生榭幹基部の遺伝子発現強度を示す図で ある。榭幹基部の師部側、および木部側より抽出した 2種の mRNAを铸型に合成した 各 cRNAを、各々 2種の蛍光色素（cy3.cy5)により標識し、オリゴマイクロアレイ解析に おけるプローブとして用い、ハイブリダィゼーシヨンを行った。スキャンして得られた画 像から蛍光強度を算出し、ロゼッタ社製の解析ソフトウェア (Luminator Ver. 1.0)にて 解析し、反復実験全てを統計的信頼度 99.999%にて統合し、ユーカリ榭幹基部にお ける主要遺伝子群の発現相対強度を表した。図中 2本の検量線のうち上の線付近ま たはその上の領域にある + (黒）は木繊維形成部位で発現上昇が有意に認められるも の、 2本の検量線のほぼ中間にある + (薄いグレー）は発現減少が有意に認められる もの、 2本の検量線のうち下の線付近またはその下の領域にある + (濃いグレー）は 発現に変化が認められないものを示す。 [0251] [Fig. 1] A graph showing the gene expression intensity of the 5-year-old ginger stem base of Eucalyptus camaldorensis. Probes for oligo microarray analysis by labeling each cRNA, which is synthesized from two types of mRNA extracted from the phloem side of the trunk base and the xylem side, in a cage shape with two fluorescent dyes (cy3.cy5) each And used for hybridization. The fluorescence intensity is calculated from the image obtained by scanning and analyzed with Rosetta's analysis software (Luminator Ver. 1.0), and all repeated experiments are integrated with a statistical reliability of 99.999%. The relative intensity of expression of major gene groups at the base is shown. In the figure, + (black) in the vicinity of or above the upper part of the two calibration curves shows a significant increase in expression at the wood fiber formation site. + (Light gray) in the middle of the two calibration curves shows a significant decrease in expression, and the two calibration curves are near or below the lower line + (dark gray) Indicates that there is no change in expression.
圆 2]ユーカリ属カマルドレンシス種の 5年生榭幹中央部の遺伝子発現強度を示す図 である。榭幹中央部の師部側、および木部側より抽出した 2種の mRNAを铸型に合成 した各 cRNAを、各々 2種の蛍光色素（cy3.cy5)により標識し、オリゴマイクロアレイ解 祈におけるプローブとして用い、ハイブリダィゼーシヨンを行った。スキャンして得られ た画像力 蛍光強度を算出し、ロゼッタ社製の解析ソフトウェア (Luminator Ver. 1.0) にて解析し、反復実験全てを統計的信頼度 99.999%にて統合し、ユーカリ榭幹中央 部における主要遺伝子群の発現相対強度を表した。図中 2本の検量線のうち上の線 付近またはその上の領域にある + (黒）は木繊維形成部位で発現上昇が有意に認め られるもの、 2本の検量線のほぼ中間にある + (薄いグレー）は発現減少が有意に認 められるもの、 2本の検量線のうち下の線付近またはその下の領域にある + (濃いグ レー）は発現に変化が認められないものを示す。 [2] It is a graph showing the gene expression intensity in the middle part of the 5-year-old trunk of the Eucalyptus genus Camaldrensis. In the oligo microarray praying, each cRNA synthesized in the cocoon shape from the phloem side and xylem side of the trunk trunk is labeled with two fluorescent dyes (cy3.cy5). Using as a probe, hybridization was performed. Image power obtained by scanning Fluorescence intensity is calculated and analyzed with Rosetta's analysis software (Luminator Ver. 1.0). All repeated experiments are integrated with a statistical reliability of 99.999%. The expression relative intensity of the major gene group in the part was expressed. In the figure, + (black) in the vicinity of or above the upper two of the two calibration curves shows a significant increase in expression at the wood fiber formation site, and is almost in the middle of the two calibration curves + (Light gray) indicates significantly decreased expression, and + (dark gray) in the vicinity of or below the lower line of the two calibration curves indicates no change in expression. .
圆 3]ユーカリ属グロブルス種榭幹の遺伝子発現強度を示す図である。榭幹の師部 側、および木部側より抽出した 2種の mRNAを铸型に合成した各 cRNAを、各々 2種の 蛍光色素（cy3.cy5)により標識し、オリゴマイクロアレイ解析におけるプローブとして用 い、ハイブリダィゼーシヨンを行った。スキャンして得られた画像力 蛍光強度を算出 し、ロゼッタ社製の解析ソフトウェア (Luminator Ver. 1.0)にて解析し、反復実験全てを 統計的信頼度 99.999%にて統合し、グロブルス種榭幹における主要遺伝子群の発現 相対強度を表した。図中 2本の検量線のうち上の線付近またはその上の領域にある + (黒）は木繊維形成部位で発現上昇が有意に認められるもの、 2本の検量線のほぼ 中間にある + (薄いグレー）は発現減少が有意に認められるもの、 2本の検量線のうち 下の線付近またはその下の領域にある + (濃いグレー）は発現に変化が認められな いものを示す。 圆 3] It is a figure showing the gene expression intensity of Eucalyptus globulis spp. Each cRNA synthesized from the phloem side and xylem side of the trunk is synthesized in a cage shape and labeled with two fluorescent dyes (cy3.cy5) for use as probes in oligo microarray analysis. I did a hybridization. Image power obtained by scanning Fluorescence intensity is calculated and analyzed with Rosetta analysis software (Luminator Ver. 1.0), and all repeated experiments are integrated with a statistical reliability of 99.999%. The relative intensity of the expression of major genes in In the figure, + (black) in the vicinity of or above the upper line of the two calibration curves shows a significant increase in expression at the wood fiber formation site, almost in the middle of the two calibration curves + (Light gray) indicates a significant decrease in expression, and + (dark gray) in the vicinity of or below the two lines of the two calibration curves indicates no change in expression.
[図 4]木繊維形成に関わる遺伝子群のプロモーター活性を示す図である。 Aは、パー ティクルガン導入用ターゲットとなる木部形成組織ブロックを切除した榭幹を示す。 B は、パーティクルガンによる遺伝子導入後に、組織ィ匕学的 GUSアツセィした木部形成 組織ブロックを示す。 Cと Dは、 GUS遺伝子が発現した青いスポット部分（〇で囲った 中心部分)の拡大写真である。 FIG. 4 is a graph showing the promoter activity of genes involved in wood fiber formation. A shows the trunk from which the xylem-forming tissue block that is the target for particle gun introduction was excised. B shows the formation of xylem by histological GUS assembly after gene transfer by particle gun. Indicates an organization block. C and D are enlarged photographs of the blue spot where the GUS gene was expressed (the central part surrounded by a circle).
[図 5-a]木繊維形成に関わる細胞壁形成遺伝子群のプロモーターと転写因子群の相 互作用（結合）を示す図である。各グラフは、 MYB転写因子と 4種のプロモーターとの 解析結果を示す。各グラフの横軸は 2本鎖 DNAプローブの番号を、縦軸はコントロー ル (プローブと空ベクター反応液）に対する並進時間の伸び率を％で表している。各 グラフ横軸のプローブ No.の小さい方がプロモーター 5'側上流域であり、大きい方が 転写開始点に近 、プロモーター 3 '側下流域である。※印は相互作用が認められた プローブを表している。  FIG. 5-a is a diagram showing the interaction (binding) between the promoter of the cell wall-forming gene group involved in tree fiber formation and the transcription factor group. Each graph shows the analysis results of the MYB transcription factor and the four promoters. In each graph, the horizontal axis represents the number of the double-stranded DNA probe, and the vertical axis represents the percentage increase in translation time relative to the control (probe and empty vector reaction solution). The smaller probe number on the horizontal axis of each graph is the upstream region on the 5 ′ side of the promoter, and the larger one is closer to the transcription start point and the downstream region on the 3 ′ side of the promoter. * Indicates a probe that has been recognized to interact.
[図 5-b]木繊維形成に関わる細胞壁形成遺伝子群のプロモーターと転写因子群の相 互作用（結合)を示す図である。各グラフは、 HD-ZIP転写因子と 4種のプロモーター との解析結果を示す。各グラフの横軸は 2本鎖 DNAプローブの番号を、縦軸はコント ロール (プローブと空ベクター反応液）に対する並進時間の伸び率を％で表して 、る 。各グラフ横軸のプローブ No.の小さい方がプロモーター 5'側上流域であり、大きい 方が転写開始点に近 、プロモーター 3 '側下流域である。※印は相互作用が認めら れたプローブを表して!/、る。  FIG. 5-b is a diagram showing the interaction (binding) between the promoter of the cell wall-forming gene group involved in tree fiber formation and the transcription factor group. Each graph shows the results of analysis of HD-ZIP transcription factors and four promoters. In each graph, the horizontal axis represents the number of the double-stranded DNA probe, and the vertical axis represents the rate of increase in translation time relative to the control (probe and empty vector reaction solution) in%. The smaller probe number on the horizontal axis of each graph is the upstream region on the 5 ′ side of the promoter, and the larger probe number is closer to the transcription start point and the downstream region on the 3 ′ side of the promoter. The asterisk (*) indicates a probe that has been recognized to interact!
圆 6]転写因子を導入した形質転換タバコ植物体を示す図である。 Aは MYB転写因 子を導入した形質転換体で、 Bはホメォドメイン転写因子を導入した形質転換体であ る。 [6] It is a diagram showing a transformed tobacco plant into which a transcription factor has been introduced. A is a transformant introduced with a MYB transcription factor, and B is a transformant introduced with a homeodomain transcription factor.

Claims

請求の範囲 The scope of the claims
[1] 以下の（a)〜（e)の 、ずれかに記載の植物樹幹の木繊維細胞壁を形成する機能を 有するタンパク質をコードする DNA。  [1] A DNA encoding a protein having a function of forming a tree fiber cell wall of a plant trunk according to any one of the following (a) to (e):
(a)配列番号： 35〜68のいずれかに記載のアミノ酸配列力 なるタンパク質をコード する DNA  (a) DNA encoding a protein having the amino acid sequence ability described in any of SEQ ID NOs: 35 to 68
(b)配列番号： 1〜34のいずれかに記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 1-34
(c)配列番号： 35〜68のいずれかに記載のアミノ酸配列力 なるタンパク質と 50%以 上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with a protein having an amino acid sequence ability according to SEQ ID NO: 35-68
(d)配列番号： 1〜34のいずれかに記載の塩基配列力 なる DNAとストリンジェントな 条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence according to SEQ ID NO: 1-34
(e)配列番号： 35〜68のいずれかに記載のアミノ酸配列において 1または複数のァ ミノ酸が置換、欠失、付加、および/または挿入されたアミノ酸配列からなるタンパク 質をコードする DNA  (e) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 35-68
[2] 植物がユーカリである、請求項 1に記載の DNA。  [2] The DNA according to claim 1, wherein the plant is eucalyptus.
[3] 請求項 1または 2に記載の DNAの部分 DNA。  [3] A partial DNA of the DNA according to claim 1 or 2.
[4] 請求項 1〜3のいずれか〖こ記載の DNAが固定ィ匕された基板。  [4] A substrate on which the DNA according to any one of claims 1 to 3 is immobilized.
[5] 植物の榭幹の師部側および木部側において、以下の（a)〜（e)に記載の少なくとも 1 つの DNAの発現レベルを検出し、師部側および木部側における該 DNAの発現レべ ルを比較する工程を含む、植物の榭幹木部の生長状態を検査する方法。  [5] On the phloem side and xylem side of the plant trunk, the expression level of at least one DNA described in the following (a) to (e) is detected, and the DNA on the phloem side and xylem side is detected. A method for examining the growth state of a stem trunk of a plant, comprising a step of comparing the expression levels of the plants.
(a)配列番号： 35〜64または 66〜68のいずれかに記載のアミノ酸配列からなるタン パク質をコードする DNA  (a) DNA encoding a protein consisting of the amino acid sequence set forth in any of SEQ ID NOs: 35 to 64 or 66 to 68
(b)配列番号： 1〜30または 32〜34のいずれかに記載の塩基配列を含む DNA (b) SEQ ID NO: DNA comprising the nucleotide sequence set forth in any of 1-30 or 32-34
(c)配列番号： 35〜64または 66〜68のいずれかに記載のアミノ酸配列からなるタン ノ ク質と 50%以上の相同性を有するタンパク質をコードする DNA (c) DNA encoding a protein having 50% or more homology with a protein consisting of the amino acid sequence of SEQ ID NO: 35-64 or 66-68
(d)配列番号： 1〜30または 32〜34のいずれかに記載の塩基配列からなる DNAとス トリンジェントな条件下でハイブリダィズする DNA  (d) SEQ ID NO: DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence of any one of 1-30 or 32-34
(e)配列番号： 35〜64または 66〜68のいずれかに記載のアミノ酸配列において 1ま たは複数のアミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配列か らなるタンパク質をコードする DNA (e) SEQ ID NO: 35-64 or 66-68, in which one or more amino acids are substituted, deleted, added, and Z or inserted amino acid sequence DNA encoding the protein
異なる 2つの榭幹木部において、以下の（a)〜（e)に記載の少なくとも 1つの DNAの 発現レベルを検出し、異なる 2つの榭幹木部における該 DNAの発現レベルを比較す る工程を含む、異なる 2つの榭幹木部の生長状態を検査する方法。 A step of detecting the expression level of at least one DNA described in the following (a) to (e) in two different trunk trunks and comparing the expression level of the DNA in two different trunk trunks A method for inspecting the growth status of two different trunks.
(a)配列番号： 35〜40、 42、 48、 50、 51、 53〜56、 58、 59、 62、 64、 66、または 6 7のいずれかに記載のアミノ酸配列力 なるタンパク質をコードする DNA  (a) DNA encoding the protein having the amino acid sequence ability according to any one of SEQ ID NOs: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62, 64, 66, or 67
(b)配列番号： 1〜6、 8、 14、 16、 17、 19〜22、 24、 25、 28、 30、 32、または 33の Vヽずれかに記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence of SEQ ID NO: 1-6, 8, 14, 16, 17, 19-22, 24, 25, 28, 30, 32, or 33
(c)配列番号： 35〜40、 42、 48、 50、 51、 53〜56、 58、 59、 62、 64、 66、または 6 (c) SEQ ID NO: 35-40, 42, 48, 50, 51, 53-56, 58, 59, 62, 64, 66, or 6
7の 、ずれかに記載のアミノ酸配列力 なるタンパク質と 50%以上の相同性を有する タンパク質をコードする DNA DNA encoding a protein having 50% or more homology with a protein having the amino acid sequence ability described in 7
(d)配列番号： 1〜6、 8、 14、 16、 17、 19〜22、 24、 25、 28、 30、 32、または 33の いずれかに記載の塩基配列力 なる DNAとストリンジヱントな条件下でノヽイブリダィズ する DNA  (d) SEQ ID NOs: 1 to 6, 8, 14, 16, 17, 19 to 22, 24, 25, 28, 30, 32, or 33 Noisy Breeding DNA
(e)配列番号： 35〜40、 42、 48、 50、 51、 53〜56、 58、 59、 62、 64、 66、または 6 7のいずれかに記載のアミノ酸配列において 1または複数のアミノ酸が置換、欠失、 付加、および Zまたは挿入されたアミノ酸配列力もなるタンパク質をコードする DNA 植物の榭幹の師部側および木部側において、以下の（a)〜（e)に記載の少なくとも 1 つの DNAの発現レベルを検出し、師部側および木部側における該 DNAの発現レべ ルを比較する工程を含む、植物の榭幹木部の生長状態を検査する方法。  (e) SEQ ID NOs: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62, 64, 66, or 67, wherein one or more amino acids are At least one of the following (a) to (e) on the phloem and xylem side of the trunk of a DNA plant that encodes a protein that also has substitution, deletion, addition, and Z or inserted amino acid sequence ability A method for examining the growth status of a stem xylem of a plant, comprising the step of detecting the expression level of two DNAs and comparing the expression level of the DNA on the phloem side and the xylem side.
(a)配列番号： 35〜40、 42、 43, 45〜48、 50、 51、 53〜59、 61、 62、 64, 66,ま たは 67のいずれかに記載のアミノ酸配列力 なるタンパク質をコードする DNA (a) SEQ ID NO: 35-40, 42, 43, 45-48, 50, 51, 53-59, 61, 62, 64, 66, or 67 DNA to encode
(b)配列番号： 1〜6、 8、 9、 11〜14、 16、 17、 19〜25、 27、 28、 30、 32、または 3 3の!、ずれかに記載の塩基配列を含む DNA (b) SEQ ID NO: 1 to 6, 8, 9, 11 to 14, 16, 17, 19 to 25, 27, 28, 30, 32, or 33!
(c)配列番号： 35〜40、 42、 43, 45〜48、 50、 51、 53〜59、 61、 62、 64, 66,ま たは 67のいずれかに記載のアミノ酸配列力 なるタンパク質と 50%以上の相同性を 有するタンパク質をコードする DNA  (c) SEQ ID NO: 35 to 40, 42, 43, 45 to 48, 50, 51, 53 to 59, 61, 62, 64, 66, or 67 DNA encoding a protein with 50% or more homology
(d)配列番号： 1〜6、 8、 9、 11〜14、 16、 17、 19〜25、 27、 28、 30、 32、または 3 3のいずれかに記載の塩基配列力もなる DNAとストリンジヱントな条件下でノヽイブリダ ィズする DNA (d) SEQ ID NO: 1-6, 8, 9, 11-14, 16, 17, 19-25, 27, 28, 30, 32, or 3 DNA that also has a base sequence ability as described in any of the above 3 and DNA that is hybridized under stringent conditions
(e)配列番号： 35〜40、 42、 43, 45〜48、 50、 51、 53〜59、 61、 62、 64, 66,ま たは 67のいずれかに記載のアミノ酸配列において 1または複数のアミノ酸が置換、欠 失、付加、および Zまたは挿入されたアミノ酸配列からなるタンパク質をコードする DN A  (e) SEQ ID NO: 35-40, 42, 43, 45-48, 50, 51, 53-59, 61, 62, 64, 66, or 67 DN A that encodes a protein consisting of amino acid sequences with substitutions, deletions, additions, and Z or inserted amino acids
[8] 異なる 2つの榭幹木部において、以下の（a)〜（c)に記載の少なくとも 1つの DNAの 発現レベルを検出し、異なる 2つの榭幹木部における該 DNAの発現レベルを比較す る工程を含む、異なる 2つの榭幹木部の生長状態を検査する方法。  [8] The expression level of at least one DNA described in the following (a) to (c) is detected in two different trunk trunks, and the expression level of the DNA in two different trunk trunks is compared. This is a method for inspecting the growth status of two different trunks.
(a)配列番号： 45、 46、 51、 55、または 56のいずれかに記載のアミノ酸配列からなる タンパク質をコードする DNA  (a) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56
(b)配列番号： 11、 12、 17、 21、または 22のいずれかに記載の塩基配列を含む DN (b) DN containing the nucleotide sequence set forth in any of SEQ ID NOS: 11, 12, 17, 21, or 22
A A
(c)配列番号： 45、 46、 51、 55、または 56のいずれかに記載のアミノ酸配列からなる タンパク質と 50%以上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56
(d)配列番号： 11、 12、 17、 21、または 22のいずれかに記載の塩基配列からなる D NAとストリンジェントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 11, 12, 17, 21, or 22
(e)配列番号： 45、 46、 51、 55、または 56のいずれかに記載のアミノ酸配列におい て 1または複数のアミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配 列からなるタンパク質をコードする DNA  (e) consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56 DNA encoding the protein
[9] 請求項 1または 2に記載の DNAによりコードされるタンパク質。  [9] A protein encoded by the DNA of claim 1 or 2.
[10] 以下の（a)〜（c)の!、ずれかの塩基配列からなるプロモーター DNA。  [10] A promoter DNA consisting of any one of the following (a) to (c)!
(a)配列番号： 93〜： LOOのいずれかに記載の塩基配列  (a) SEQ ID NO: 93-: The nucleotide sequence according to any one of LOO
(b) (a)の塩基配列において、 1又は数個の塩基が欠失、置換又は付加された塩基 配列からなり、かつ、（a)の DNAからなるプロモーターと実質的に同一のプロモーター としての機能を有する塩基配列  (b) In the base sequence of (a), it consists of a base sequence in which one or several bases are deleted, substituted or added, and is substantially the same promoter as the promoter composed of the DNA of (a). Functional nucleotide sequence
(c) (a)の塩基配列の一部を含み、かつ、（a)の DNAからなるプロモーターと実質的 に同一のプロモーターとしての機能を有する塩基配列 [11] さらに、植物の木繊維形成組織に特異的発現に関与する転写因子を認識する塩基 配列を機能的に結合させた、請求項 10に記載のプロモーター DNA。 (c) a base sequence that includes a part of the base sequence of (a) and has a function as a promoter substantially the same as the promoter comprising the DNA of (a) [11] The promoter DNA according to claim 10, further comprising a base sequence that recognizes a transcription factor involved in specific expression in a plant fiber-forming tissue.
[12] 以下の（a)〜（e)のいずれかに記載の DNA。 [12] The DNA according to any of (a) to (e) below.
(a)請求項 1に記載の DNAの転写産物と相補的なアンチセンス RNAをコードする DN A  (a) DNA encoding antisense RNA complementary to the transcription product of DNA according to claim 1
(b)請求項 1に記載の DNAの転写産物を特異的に開裂するリボザィム活性を有する RNAをコードする DNA  (b) a DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcript of the DNA of claim 1
(c) RNAi効果により、請求項 1に記載の DNAの発現を抑制する RNAをコードする DN A  (c) DNA encoding RNA that suppresses the expression of DNA according to claim 1 due to RNAi effect
(d)共抑制効果により、請求項 1に記載の DNAの発現を抑制する RNAをコードする D NA  (d) DNA encoding RNA that suppresses the expression of DNA according to claim 1 due to a co-suppression effect
(e)請求項 1に記載の DNAの転写産物に対してドミナントネガティブな形質を有する タンパク質をコードする DNA  (e) DNA encoding a protein having a dominant negative trait for the transcript of the DNA of claim 1
[13] 以下の（a)〜（e)の!、ずれかに記載の植物の転写因子をコードする DNA。  [13] A DNA encoding a plant transcription factor described in any one of (a) to (e) below.
(a)配列番号： 112〜 122の 、ずれかに記載のアミノ酸配列力もなるタンパク質をコ ードする DNA  (a) DNA encoding a protein having an amino acid sequence ability described in any one of SEQ ID NOs: 112 to 122
(b)配列番号：101〜： L 11の!、ずれかに記載の塩基配列を含む DNA  (b) SEQ ID NO: 101-: DNA comprising the base sequence described in L11!
(c)配列番号： 112〜 122の 、ずれかに記載のアミノ酸配列力もなるタンパク質と 50 %以上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein of SEQ ID NO: 112 to 122, which also has an amino acid sequence ability described in any one of
(d)配列番号： 101〜111のいずれかに記載の塩基配列からなる DNAとストリンジ ントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 101-111
(e)配列番号： 112〜 122の 、ずれかに記載のアミノ酸配列にお 、て 1または複数の アミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配列力 なるタンパ ク質をコードする DNA  (e) In the amino acid sequence according to any one of SEQ ID NOS: 112 to 122, one or a plurality of amino acids are substituted, deleted, added, and encoded by a protein having an amino acid sequence ability by Z or insertion DNA
[14] 植物がユーカリである、請求項 13に記載の DNA。  [14] The DNA of claim 13, wherein the plant is eucalyptus.
[15] 請求項 13または 14に記載の DNAの部分 DNA。  [15] A partial DNA of the DNA according to claim 13 or 14.
[16] 請求項 13〜 15のいずれかに記載の DNAが固定ィ匕された基板。  [16] A substrate on which the DNA according to any one of claims 13 to 15 is immobilized.
[17] 植物の榭幹の師部側および木部側において、以下の（a)〜（e)に記載の少なくとも 1 つの DNAの発現レベルを検出し、師部側および木部側における該 DNAの発現レべ ルを比較する工程を含む、植物の榭幹木部の生長状態を検査する方法。 [17] On the phloem side and xylem side of the plant trunk, at least 1 described in (a) to (e) below: A method for examining the growth status of a stem xylem of a plant, comprising the step of detecting the expression level of two DNAs and comparing the expression level of the DNA on the phloem side and the xylem side.
(a)配列番号： 112〜 118または 120〜 122の!、ずれかに記載のアミノ酸配列からな るタンパク質をコードする DNA  (a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 112-118 or 120-122
(b)配列番号： 101〜107または 109〜111のいずれかに記載の塩基配列を含む D NA  (b) SEQ ID NO: 101-107 or 109-111
(c)配列番号： 112〜 118または 120〜 122の!、ずれかに記載のアミノ酸配列からな るタンパク質と 50%以上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with a protein consisting of the amino acid sequence of SEQ ID NO: 112-118 or 120-122
(d)配列番号： 101〜107または 109〜111のいずれかに記載の塩基配列からなる DNAとストリンジェントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 101-107 or 109-111
(e)配列番号： 112〜 118または 120〜 122の!ヽずれかに記載のアミノ酸配列にお いて 1または複数のアミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸 配列からなるタンパク質をコードする DNA  (e) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence of SEQ ID NO: 112-118 or 120-122. DNA encoding
[18] 異なる 2つの榭幹木部において、以下の（a)〜（e)に記載の少なくとも 1つの DNAの 発現レベルを検出し、異なる 2つの榭幹木部における該 DNAの発現レベルを比較す る工程を含む、異なる 2つの榭幹木部の生長状態を検査する方法。  [18] In two different trunk trunks, detect the expression level of at least one DNA described in (a) to (e) below, and compare the expression level of the DNA in two different trunk trunks This is a method for inspecting the growth status of two different trunks.
(a)配列番号： 113または 114に記載のアミノ酸配列力もなるタンパク質をコードする DNA  (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 113 or 114
(b)配列番号： 102または 103に記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 102 or 103
(c)配列番号： 113または 114に記載のアミノ酸配列力もなるタンパク質と 50%以上 の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in SEQ ID NO: 113 or 114
(d)配列番号： 102または 103に記載の塩基配列からなる DNAとストリンジ ントな条 件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 102 or 103
(e)配列番号： 113または 114に記載のアミノ酸配列にお 、て 1または複数のアミノ酸 が置換、欠失、付加、および/または挿入されたアミノ酸配列力 なるタンパク質をコ ードする DNA  (e) DNA encoding a protein having an amino acid sequence ability in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence set forth in SEQ ID NO: 113 or 114
[19] 植物の榭幹の師部側および木部側において、以下の（a)〜（e)に記載の少なくとも 1 つの DNAの発現レベルを検出し、師部側および木部側における該 DNAの発現レべ ルを比較する工程を含む、植物の榭幹木部の生長状態を検査する方法。 [19] The expression level of at least one DNA described in the following (a) to (e) is detected on the phloem side and xylem side of the trunk of the plant, and the DNA on the phloem side and xylem side is detected. Expression level A method for inspecting the growth state of a stem trunk of a plant, comprising a step of comparing
(a)配列番号： 113〜 117の 、ずれかに記載のアミノ酸配列力 なるタンパク質をコ ードする DNA  (a) DNA encoding a protein having the amino acid sequence ability described in any one of SEQ ID NOS: 113 to 117
(b)配列番号： 102〜106のいずれかに記載の塩基配列を含む DNA  (b) DNA comprising the nucleotide sequence according to any one of SEQ ID NOs: 102 to 106
(c)配列番号： 113〜 117の 、ずれかに記載のアミノ酸配列力もなるタンパク質と 50 %以上の相同性を有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein of SEQ ID NO: 113 to 117 which also has an amino acid sequence ability described in any
(d)配列番号： 102〜 106のいずれかに記載の塩基配列からなる DNAとストリンジェ ントな条件下でハイブリダィズする DNA  (d) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 102 to 106
(e)配列番号： 113〜 117の 、ずれかに記載のアミノ酸配列にお 、て 1または複数の アミノ酸が置換、欠失、付加、および Zまたは挿入されたアミノ酸配列力 なるタンパ ク質をコードする DNA  (e) SEQ ID NO: 113-117, which encodes a protein having an amino acid sequence ability in which one or more amino acids are substituted, deleted, added, and Z or inserted in any one of the amino acid sequences described in any one of DNA
[20] 異なる 2つの榭幹木部において、以下の（a)〜（c)に記載の少なくとも 1つの DNAの 発現レベルを検出し、異なる 2つの榭幹木部における該 DNAの発現レベルを比較す る工程を含む、異なる 2つの榭幹木部の生長状態を検査する方法。  [20] The expression level of at least one DNA described in the following (a) to (c) is detected in two different trunk trunks, and the expression level of the DNA in two different trunk trunks is compared. This is a method for inspecting the growth status of two different trunks.
(a)配列番号： 116に記載のアミノ酸配列力もなるタンパク質をコードする DNA (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 116
(b)配列番号： 105に記載の塩基配列を含む DNA (b) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 105
(c)配列番号： 116に記載のアミノ酸配列力もなるタンパク質と 50%以上の相同性を 有するタンパク質をコードする DNA  (c) DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in SEQ ID NO: 116
(d)配列番号： 105に記載の塩基配列からなる DNAとストリンジ ントな条件下でノ、ィ ブリダィズする DNA  (d) DNA that is hybridized under stringent conditions with DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 105
(e)配列番号： 116に記載のアミノ酸配列において 1または複数のアミノ酸が置換、欠 失、付加、および Zまたは挿入されたアミノ酸配列からなるタンパク質をコードする DN A  (e) DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence set forth in SEQ ID NO: 116
[21] 請求項 13または 14に記載の DNAによりコードされるタンパク質。  [21] A protein encoded by the DNA of claim 13 or 14.
[22] 以下の（a)〜（c)の!、ずれかの塩基配列からなるプロモーター DNA。 [22] Promoter DNA consisting of any one of the following (a) to (c)!
(a)配列番号：156〜166の!、ずれかに記載の塩基配列  (a) SEQ ID NO: 156 to 166!
(b) (a)の塩基配列において、 1又は数個の塩基が欠失、置換又は付加された塩基 配列からなり、かつ、（a)の DNAからなるプロモーターと実質的に同一のプロモーター としての機能を有する塩基配列 (b) A promoter consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence of (a), and substantially the same promoter as the promoter of DNA of (a) Base sequence that functions as
(c) (a)の塩基配列の一部を含み、かつ、（a)の DNAからなるプロモーターと実質的 に同一のプロモーターとしての機能を有する塩基配列  (c) a base sequence comprising a part of the base sequence of (a) and having a function as a promoter substantially the same as the promoter comprising the DNA of (a)
[23] さらに、植物の木繊維形成組織に特異的発現に関与する転写因子を認識する塩基 配列を機能的に結合させた、請求項 22に記載のプロモーター DNA。 [23] The promoter DNA according to claim 22, further comprising a base sequence that recognizes a transcription factor involved in specific expression functionally linked to a plant fiber-forming tissue.
[24] 請求項 1〜3、 10〜15、 22または 23のいずれかに記載の DNAを有する組換えべク ター。 [24] A recombinant vector comprising the DNA of any one of claims 1-3, 10-15, 22 or 23.
[25] 請求項 24に記載のベクターを含むプラスミドを保持する微生物。  [25] A microorganism carrying a plasmid comprising the vector of claim 24.
[26] 請求項 24に記載のベクターが導入された形質転換植物細胞。  [26] A transformed plant cell into which the vector according to claim 24 has been introduced.
[27] 請求項 26に記載の形質転換植物細胞から再分化した形質転換植物体。  [27] A transformed plant regenerated from the transformed plant cell according to claim 26.
[28] 請求項 27に記載の形質転換植物体の子孫またはクローンである、形質転換植物体  [28] A transformed plant which is a descendant or clone of the transformed plant according to claim 27
[29] 請求項 27または 28に記載の形質転換植物体の繁殖材料。 [29] The propagation material for the transformed plant according to claim 27 or 28.
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