CN110373418B - Gene for regulating and controlling plant seed size and application thereof - Google Patents

Gene for regulating and controlling plant seed size and application thereof Download PDF

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CN110373418B
CN110373418B CN201910752694.7A CN201910752694A CN110373418B CN 110373418 B CN110373418 B CN 110373418B CN 201910752694 A CN201910752694 A CN 201910752694A CN 110373418 B CN110373418 B CN 110373418B
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唐晓艳
许纯珏
严维
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FRONTIER LABORATORIES OF SYSTEMS CROP DESIGN CO LTD
Shenzhen Institute of Molecular Crop Design
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Abstract

The invention discloses a gene for regulating and controlling the size of plant seeds and application thereof, belonging to the fields of plant molecular biology, biochemistry, genetics and plant breeding. The invention obtains a large mutant by EMS mutagenesis rice, and locates the mutation character related gene by SIMM method to obtain a gene DLZ for regulating and controlling plant seed size, the gene is located on chromosome 12, and the locus number is LOC_Os12g41820 (MSU login number). The mutation of the gene DLZ can lead rice to generate a large grain phenotype, the grain size of plants can be regulated and controlled by regulating the expression of the gene, and the method for obtaining large grain mutant plants and improving crops has important theoretical and practical significance for the high-yield breeding work of crops.

Description

Gene for regulating and controlling plant seed size and application thereof
Technical Field
The invention relates to the fields of plant molecular biology, biochemistry, genetics and plant breeding, in particular to a gene for regulating and controlling seed size, and more particularly relates to a nucleic acid molecule of DLZ gene for regulating and controlling seed size, a mutant thereof and application thereof in breeding.
Technical Field
Plant seeds are the most important food sources for human survival, rice, corn and wheat are three main grains in China, and the grain yield and the price influence domestic life. The current situation that the population is continuously increased and the cultivated area is continuously reduced provides a serious challenge for the grain production in China. Taking rice as an example, the rice yield is mainly determined by three factors, namely effective spike number, solid grain number per spike and grain weight, the yield per unit area is also influenced by the plant type of the rice, the factors are complex traits controlled by the interaction of multiple genes and environment, the sub-traits have correlation, and the relation among the constituent elements needs to be coordinated for improving the yield traits. According to the prior experience, the high-yield varieties can be divided into four types, namely large-spike weight, large-grain weight, multi-spike weight and comprehensive consideration. The genetic force of the effective spike number of the single plant is minimum, the effective spike number of the single plant is easy to be influenced by the environment, and the improvement of the effective spike number of the single plant is not better than the adjustment of the effective spike number of the unit area through reasonable close planting. The genetic power of the grain number per spike is moderate, and the improvement is still difficult. In contrast, grain weight is the highest genetic power among the yield components, and it is relatively easy to improve it (inspired, conception and practice of green super rice, scientific press, 2009).
The grain weight of rice is determined by two factors, namely the size of seeds, namely the volume of glumes and the development condition of endosperm. The glumes are usually shaped prior to flowering of the rice, and the size and shape of the rice grains formed by the grout after fertilization is limited by the volume of the glumes, and therefore, the volume of the glumes is a prerequisite for the size of the grain size. The description of glume shape and size is generally referred to as grain shape, and is generally expressed in terms of grain length, grain width, grain thickness, and aspect ratio. It is worth mentioning that the grain shape is not only one of important yield traits, but also a main appearance quality trait, and that there is a certain correlation between the grain shape and other quality traits of rice, such as chalkiness rate, brown rice rate, polished rice rate. Therefore, in breeding, through selecting the size and shape of seeds, not only the yield potential can be improved, but also the rice quality can be indirectly regulated and controlled.
Plant seed size is a quantitative trait controlled by multiple genes, and cloning of related genes has shown a burst trend in the last decade on the basis of research of early-stage large-scale QTL scanning and positioning. The excavation and functional research of important crop seed size regulation genes such as rice have become a great hotspot in functional genomics research. The discovery and functional analysis of the genes promote the gradual penetration of the research of the plant seed size gene regulation network, researchers begin to explore the optimal design of key gene polymerization, and theoretical guidance and material support are provided for creating design type yield breakthrough new varieties by evaluating the yield increasing effect of excellent genotype polymerization.
SIMM(Simultaneous Identification of Multiple Mutations)(Yan et al.,Simultaneous identification of multiple causal mutations in rice.Frontiers in Plant Science,2016) Is a rapid and efficient method for positioning mutant genes based on a second-generation sequencing technology. Compared with other methods, the SIMM can simultaneously identify a plurality of mutation sites related to mutation characters by taking other mutants of the same or similar sources as the background under the condition that precursors of wild genome data are not needed, has higher sensitivity and specificity, is favorable for rapidly positioning candidate functional genes, and assists in rice functional gene research and rice design breeding. In addition, SIMM can also be used for locating extreme phenotype pool QTL (quantitative trait loci, quantitative Trait Locus), effectively reducing candidate intervals and assisting in locating major QTL genes. This method can also be effectively applied to locate EMS mutant candidate functional genes of other species.
The High-resolution melting curve analysis (High-Resolution Melting Curve Analysis, HRM) is a novel gene analysis technology for forming melting curves of different forms based on the difference of single nucleotide melting temperatures, has extremely High sensitivity, can detect the difference of single bases, and has the advantages of low cost, high flux, high speed, accurate result and no limitation of detection sites. The method does not require the use of sequence specific probes, but rather uses a saturation dye to analyze the PCR reaction products. The principle is as follows: the thermal stability of double-stranded DNA is affected by its length and base composition, and sequence changes can lead to changes in the melting behavior of double-stranded DNA during warming. Because the fluorescent dye can only be embedded and combined on the double-stranded DNA, the real-time PCR technology is utilized to visually display the difference existing in the PCR products in a mode of generating melting curves with different shapes by detecting the change of fluorescent signal values in the melting process of the double-stranded DNA in real time. Meanwhile, genotyping or classifying of the test population based on the melting curves of different shapes can be realized by means of professional analysis software.
According to the invention, through EMS mutagenesis of indica rice variety "Huanghuazhan", a large-grain mutant controlled by a single recessive nuclear gene is obtained through screening, and then phenotype identification, genetic analysis and genetic background identification are carried out on the mutant, and a seed size regulating gene DLZ is successfully positioned and cloned by utilizing a SIMM method, an HRM technology and gene information analysis, the gene is positioned on chromosome 12, the locus number is LOC_Os12g41820 (MSU login number), and the mutation of the gene can lead rice to generate a large-grain phenotype, so that the method can be applied to controlling the size of crop grains. The invention is beneficial to improving crop yield and quality, provides gene resources and technical support for cultivating new rice varieties with large grain weight, and has important significance and application value for improving crop agronomic characters and high-yield molecular breeding work.
Disclosure of Invention
All references mentioned herein are incorporated herein by reference.
Unless defined to the contrary, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless indicated to the contrary, the techniques used or referred to herein are standard techniques well known to those of ordinary skill in the art. The materials, methods, and examples are illustrative only and not intended to be limiting.
The invention provides a gene DLZ (large grain occupation, da Li Zhan) for regulating and controlling the size of seeds, the gene is positioned on chromosome 12, the gene locus number of the gene in rice is LOC_Os12g41820 (MSU login number, refer to Rice Genome Annotation Project, http:// rice. Plant biology. MSU. Edu /), and the mutation of the gene can make the seed grain of plants containing the mutation larger.
Since the same gene often has single nucleotide polymorphism between different varieties, that is, the nucleotide sequence of the same gene often has individual base differences, but the number of rice varieties is large, the inventors cannot enumerate one by one, so the present invention only provides sequences of two varieties representative in indica rice and japonica rice. Specifically, the nucleotide sequence of the rice DLZ gene is selected from one of the sequences of the following groups:
(a) A nucleotide sequence shown as a gene LOC_Os12g41820 for regulating the size of plant seeds;
(b) As set forth in SEQ ID NO: 1. 2, 20 or 21;
(c) The coding amino acid sequence is shown as SEQ ID NO: 3. 55, 56, 57, 58, 59, 60 or 61;
(d) A DNA sequence capable of hybridizing under stringent conditions to the DNA of the sequences set forth in (a) - (c); or (e) a DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences of (a) - (d) and having the function of regulating plant seed size; or (b)
(F) A DNA sequence complementary to any one of the sequences of (a) - (e).
The DLZ gene for regulating the seed size can be isolated from various plants. It will be appreciated by those skilled in the art that the seed size controlling gene of the present invention also includes homologous genes which are highly homologous to the nucleotide sequence or protein sequence of the DLZ gene and have the same seed size controlling function. The homologous gene includes a DNA sequence capable of hybridizing under stringent conditions to the nucleotide sequence of the DLZ gene disclosed in the present invention. As used herein, "stringent conditions" are well known and include, for example, hybridization in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH 6.4) and 1mM EDTA, preferably at a temperature of 53℃to 60℃for a period of preferably 12 to 16 hours, followed by washing with a washing solution containing 0.5 XSSC and 0.1% SDS, preferably at a temperature of 62℃to 68℃for a period of 15 to 60 minutes.
Homologous genes also include DNA sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to the sequences shown in the DLZ genes disclosed herein and having seed size control functions, and can be isolated from any plant. The percentage of sequence similarity may be obtained by well known bioinformatics algorithms, including Myers and Miller algorithms, needleman-Wunsch global alignment, smith-Waterman local alignment, pearson and Lipman similarity search, karlin and Altschul algorithms, as is well known to those skilled in the art.
The gene sequences described herein can be isolated from any plant, including but not limited to Brassica, maize, wheat, sorghum, crambe, white mustard, castor bean, sesame, cotton seed, linseed, soybean, arabidopsis, phaseolus, peanut, alfalfa, oat, rapeseed, barley, oat, rye (Rye), millet, milo, triticale, single grain wheat, spelt (Spelt), double grain wheat, flax, granola (GRAMMA GRASS), friction grass, pseudomilo, fescue, perennial wheat, sugarcane, blackberry, papaya, banana, safflower, oil palm, melon, apple, cucumber, dendrobe, sword, chrysanthemum, liliaceae, cotton, eucalyptus, sunflower, sugar beet, coffee, ornamental plants, pine and the like. Preferably, plants include corn, soybean, safflower, mustard, wheat, barley, rye, rice, cotton and sorghum.
The invention provides a method for regulating and controlling the size of plant seeds, which influences the size of the plant seeds by influencing the expression level of DLZ genes provided by the invention. The effect on plant seed size refers to the change in seed size of the plant, such as a phenotype that results in large grains, by decreasing the expression level of the DLZ gene. Specifically, depending on the specific application requirements, the expression level of the DLZ gene in plants can be influenced by various methods, thereby achieving the effect of regulating the seed size. More specifically, the regulation of the expression level of the DLZ gene can be carried out using any of a number of means available to those of ordinary skill in the art, such as by mutation, mutagenesis, transfer of antisense genes, co-suppression or introduction of hairpin structures, etc., which can be used to disrupt the normal expression of the DLZ gene and thereby obtain a plant with a larger seed.
The present invention also provides a method for obtaining large mutant materials of DLZ gene by mutating the DLZ gene endogenous to rice or mutating the nucleotide sequence of the gene highly homologous thereto, so that the action pathway of regulating seed size of the plant is changed. The nucleotide sequence of the DLZ gene is shown as SEQ ID NO: 1.2, 20 or 21, the amino acid sequence of the DLZ gene is shown as SEQ ID NO: 3. 55, 56, 57, 58, 59, 60 or 61. The "mutation" includes, but is not limited to, a method such as a gene mutation caused by a physical or chemical method, a chemical method including a mutation caused by a mutagen such as EMS, a point mutation, a DNA deletion or insertion mutation, a gene silencing means such as RNAi, or a gene site-directed mutation method including, but not limited to, a zFN site-directed mutation method, a TALEN site-directed mutation method, and/or a gene editing method such as CRISPR/Cas 9.
The invention also provides an application method of the DLZ large grain mutant material, which is characterized in that the mutant material is caused by mutation of a nucleotide sequence of a DLZ gene, a plant containing the mutant DLZ gene has a phenotype of large grain seeds, wherein the nucleotide sequence of the DLZ gene is preferably shown as SEQ ID NO: 1. 2, 20 or 21. Specifically, the nucleotide sequence after mutation is shown as SEQ ID NO:4, the amino acid sequence is shown as SEQ ID NO:5, in the macromutant, a two base mutation, in particular from AAA to TTA, occurs on the fourth exon of the DLZ gene, resulting in the sequence of SEQ ID NO: mutation of amino acid 321 of 3 protein from Lys to Leu results in variation of both the obtained transcript and protein product, thus leading to plant with large seed phenotype. Those skilled in the art will appreciate that the nucleotide sequence of SEQ ID NO:4 constructing a plant expression vector, and carrying out plant transformation to obtain a novel transgenic large-grain mutant material. Applications of the mutant materials include, but are not limited to, applications in cross breeding, and more particularly applications including, but not limited to, breeding of plant varieties or lines with increased seed size, breeding of plant varieties or lines with decreased seed size, molecular markers for identifying large and small varieties of crops, and the like.
The invention also provides application of the expression cassette in regulating the size of plant seeds, wherein the expression cassette contains a DNA sequence of a DLZ gene for regulating the size of plant seeds, and the nucleotide sequence of the gene for regulating the size of plant seeds is selected from one of the following sequences:
(a) A nucleotide sequence shown as a gene LOC_Os12g41820 for regulating the size of plant seeds;
(b) As set forth in SEQ ID NO: 1. 2, 20 or 21;
(c) The coding amino acid sequence is shown as SEQ ID NO: 3. 55, 56, 57, 58, 59, 60 or 61;
(d) A DNA sequence capable of hybridizing under stringent conditions to the DNA of the sequences set forth in (a) - (c); or (e) a DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences of (a) - (d) and having the function of regulating plant seed size; or (b)
(F) A DNA sequence complementary to any one of the sequences of (a) - (e).
Specifically, the seed size controlling gene in the above expression cassette is further operably linked to a promoter that drives expression thereof, and the promoter in the construct may be a natural promoter or a substituted promoter that will drive expression of the linked nucleotide sequence in a plant. Promoters in the expression cassette include, but are not limited to, constitutive expression promoters, inducible promoters, tissue-specific expression promoters, space-time-specific expression promoters, and the like. The gene expression of the constitutive promoter has no tissue and time specificity, and the external factors hardly affect the exogenous gene expression started by the constitutive promoter. Such constitutive promoters include, but are not limited to, caMV35S, FMV S, the rice Actin (Actin 1) promoter, the maize Ubiquitin (Ubiquitin) promoter, and the like. The tissue specific promoter has the characteristics of enhancer and silencer besides the due general promoter elements, and has the advantages of promoting the expression of genes at specific tissue positions of plants, avoiding unnecessary expression of exogenous genes and further saving the overall energy consumption of the plants. The inducible promoter disclosed by the invention refers to a promoter capable of greatly improving the transcription level of genes under the stimulation of certain specific physical or chemical signals, and the inducible promoters which have been separated at present comprise but are not limited to stress inducible expression promoters, light inducible expression promoters, heat inducible expression promoters, wound inducible expression promoters, fungus inducible expression promoters, symbiotic bacteria inducible expression promoters and the like. The tissue-specific promoters described herein include, but are not limited to, LTP2 seed-specific expression promoters, END2 seed-specific expression promoters, aleurone layer-specific expression promoters, and the like.
Other components may also be included in the above-described expression cassette, depending primarily on the purpose and use of the vector construction, and may further include, for example, selectable marker genes, targeting or regulatory sequences, stabilizing or targeting sequences, introns, and the like. Other components may also be included in the above constructs, depending primarily on the purpose and use of the vector construction, and may further include, for example, selectable marker genes, targeting or regulatory sequences, stabilizing or targeting sequences, introns, and the like. The expression cassette will also include transcription and translation terminators at the 3' end of the heterologous nucleotide sequence of interest that are functional in plants. The terminator may be the self terminator of the DLZ gene provided by the present invention, or may be a terminator derived from an external source, such as a nopaline synthase or octopine synthase termination region.
Where it is desired to direct the expression product of a heterologous nucleotide sequence to a particular organelle, such as a plastid, a amyloplast, or to the endoplasmic reticulum, or in the case of cell surface or extracellular secretion, the expression cassette may further comprise a nucleotide sequence for encoding a transit peptide. Such transit peptides are well known in the art and include, but are not limited to, small subunits of Rubisco, plant EPSP synthase, maize Brittle-1, chloroplast transit peptide, and the like.
In preparing the expression cassette, various DNA fragments may be manipulated to provide the DNA sequence in the appropriate orientation, or in the correct reading frame. To this end, adaptors or linkers may be used to ligate the DNA fragments, or further include other manipulations to provide convenient restriction sites, and the like.
The expression cassette of the present invention may further comprise a screening gene which can be used to screen plants, plant tissue cells or vectors containing the expression cassette. Such screening genes include, but are not limited to, antibiotic resistance genes, or herbicide resistance genes, or fluorescent protein genes, and the like. Specifically, the screening genes include, but are not limited to: chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, qamycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, etc.
Further, the constructs provided herein may also include a selectable marker gene for selecting transformed cells or tissues. The selectable marker genes include genes that confer antibiotic resistance or resistance to herbicides. Suitable selectable marker genes include, but are not limited to: chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, zithromycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene. The selection marker gene can also be red fluorescent gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, anthocyanin p1 and other genes.
The expression cassette or vector provided by the present invention may be inserted into a plasmid, cosmid, yeast artificial chromosome, bacterial artificial chromosome, or any other vector suitable for transformation into a host cell. Preferred host cells are bacterial cells, in particular bacterial cells for cloning or storing polynucleotides, or for transforming plant cells, such as E.coli, agrobacterium tumefaciens and Agrobacterium rhizogenes. When the host cell is a plant cell, the expression cassette or vector may be inserted into the genome of the transformed plant cell. The insertion may be a localized or random insertion. Preferably, the insertion is achieved by, for example, homologous recombination. Alternatively, the expression cassette or vector may remain extrachromosomal. The expression cassette or vector of the invention may be present in the nucleus, chloroplast, mitochondria and/or plastid of a plant cell. Preferably, the expression cassette or vector of the invention is inserted into the chromosomal DNA of the plant cell nucleus.
The nucleotide sequence and promoter sequence or expression cassette of the DLZ gene provided by the present invention may be inserted into a vector, plasmid, yeast artificial chromosome, bacterial artificial chromosome or any other vector suitable for transformation into a host cell. Preferred host cells are bacterial cells, in particular bacterial cells for cloning or storing polynucleotides, or for transforming plant cells, such as E.coli, agrobacterium tumefaciens and Agrobacterium rhizogenes. When the host cell is a plant cell, the expression cassette or vector may be inserted into the genome of the transformed plant cell. The insertion may be a localized or random insertion.
The method for transferring the nucleotide sequence, the vector or the expression cassette into a plant or introducing the nucleotide sequence, the vector or the expression cassette into the plant or transforming the plant refers to transferring the nucleotide sequence, the vector or the expression cassette into a receptor cell or a receptor plant by a conventional transgenic method. Any transgenic method known to those skilled in the art of plant biotechnology can be used to transform a recombinant expression vector into a plant cell to produce a transgenic plant of the invention. Transformation methods may include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, and microinjection. The transformation method also includes agrobacterium-mediated plant transformation methods and the like.
The invention provides a method for producing a hybrid plant for controlling the size of seeds, which is characterized by comprising the following steps:
(a) Constructing an expression cassette provided by the invention;
(b) Introducing the expression cassette obtained in step (a) into a plant cell;
(c) Regenerating the transgenic plant; and
(D) Selecting transgenic plants; and
(E) Optionally, proliferating the plant obtained in step (d) to obtain offspring.
The transgenic plants of the invention are prepared using transformation methods known to those skilled in the art of plant biotechnology. Any method can be used to transform a recombinant expression vector into a plant cell to produce a transgenic plant of the invention. Transformation methods may include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, microinjection, and the like. In specific embodiments of the present invention, the present invention uses Agrobacterium-based transformation Techniques (see Horsch RB et al (1985) Science 225:1229;White FF,Vectors for Gene Transfer in Higher Plants,Transgenic Plants, volume 1, ENGINEERING AND. Utilization, ACADEMIC PRESS,1993, pp.15-38; jenes B et al, techniques for GENE TRANSFER, TRANSGENIC PLANTS, volume 1, ENGINEERING AND. Utilization, ACADEMIC PRESS,1993, pp.128-143, etc.). Agrobacterium strains (e.g., agrobacterium tumefaciens or agrobacterium rhizogenes) contain plasmids (Ti or Ri plasmids) and T-DNA elements that are transferred to plants after transfection with agrobacterium and the T-DNA is integrated into the genome of the plant cells. The T-DNA may be located on the Ri-plasmid or the Ti-plasmid or independently contained in a so-called binary vector. Methods of agrobacterium-mediated transformation are described, for example, in. Agrobacterium-mediated transformation is most suitable for dicotyledonous plants, but also for monocotyledonous plants. Transformation of plants with agrobacterium is described, for example, in. Transformation can result in transient or stable transformation and expression. Although the nucleotide sequences of the invention can be inserted into any plant and plant cell falling into these broad categories, they are particularly suitable for use in crop plant cells.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides a rice seed size regulating gene, and the mutation of the gene can enlarge the grain of crops (such as rice), thereby increasing the crop yield and providing a new gene resource for high-yield breeding of rice.
(2) The DLZ gene provided by the invention can be used as a gene for controlling the grain size of crops and improving the yield and quality, is applied to the improvement of crop varieties, and is beneficial to breeding new rice varieties with high quality traits. Meanwhile, the DLZ gene can be also used for molecular marking technology, and is used for practical production and application services such as rice large-grain high-yield breeding and the like.
(3) The DLZ gene provided by the invention has homologous genes in a plurality of plants such as corn, sorghum and the like, and can be used for rice and other plant new variety cultivation.
Drawings
Fig. 1 is plant morphology of wild-type yellow warfare (HHZ) and large grain mutant (dlz), bar=20 cm.
Fig. 2 is the seed (a) and rice (B) morphology of yellow warrior (HHZ) and large grain mutant (dlz), bar=1 cm.
FIG. 3 is thousand kernel weight of a large mutant (dlz) hybridized F 2 generation segregating population with Huanghua (HHZ).
FIG. 4 is a diagram showing the location of the mutation site of the macromutant (dlz) by the SIMM method, wherein the red triangle indicates the position of the mutation site, the black letter indicates the base of the wild type, and the red letter indicates the base of the mutant.
FIG. 5 is a graph showing the grain width and thousand kernel weight of a recombinant and partially non-recombinant individual between three candidate genes in a large mutant (dlz) and Huanghua (HHZ) hybrid F 2 generation segregating population, wherein W represents a wild type, H represents a heterozygous mutant, and M represents a homozygous mutant.
FIG. 6 shows the expression level of DLZ gene in different tissues and organs of wild type Huanghua. DAP1 represents 1 day after fertilization.
FIG. 7 is a sequence variation (A) generated by site-directed knock-out of the DLZ gene of japonica rice middle flower 11 (ZH 11) and indica yellow Huazhan (HHZ) using CRISPR technique and the corresponding transgenic plant seed size (B), wherein the red letter indicates inserted bases and the red "-" indicates deleted bases; z series numbers represent transgenic plants with flower 11 background in japonica rice, and H series numbers represent transgenic plants with yellow Huazhan background.
FIG. 8 is a comparison of various important agronomic trait phenotypes and individual yield of wild type Huanghua (W), heterozygous mutant (H) and dlz homozygous mutant (M), wherein the different letters a, b, c represent significant differences in the data over t-test and the same letters represent insignificant differences.
FIG. 9 shows the expression level of DLZ gene and its grain width phenotype of transgenic plants produced by gene silencing of DLZ from flower 11 in japonica rice using RNAi technique, wherein gray columns represent transgenic negative plants.
FIG. 10 is a graph showing the expression level of DLZ gene, deletion of promoter fragment and grain width phenotype of transgenic plants produced by editing the DLZ gene promoter of flower 11 in japonica rice by CRISPR technique, wherein gray columns represent individual plants in which the variation in expression amount and the variation in grain width phenotype are not apparent.
FIG. 11 is a homology comparison of homologous DLZ proteins from different plants, wherein Brachypodium di represents Brachypodium distachyranum, hordeum vulgare represents barley, oryza sativa represents rice, oryza brachyant represents wild rice, panicum hallii represents millet, SETARIA ITALICA represents millet, zea mays represents maize, and Sorgum bicolor represents Sorghum.
Detailed Description
The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.
Example 1 screening of Rice macromutant (dlz)
Soaking indica yellow Huazhan seeds (M 0) in an EMS aqueous solution with the mass concentration of 0.7%, carrying out mutagenesis treatment for 12 hours, and carrying out seed setting on the seeds of the generation M 0 and then carrying out mixed harvest to obtain a mutant library (M 1). Plants from the M 1 generation seeds were used for screening during seed maturity and plants with normal plant development and significantly enlarged seeds were obtained by phenotypic observation (fig. 1, fig. 2A). The enlargement of mutant seeds is mainly due to the increase in grain width, and the grain fullness is not affected after the glume shell volume is increased, so the width of the rice grains is also significantly increased (fig. 2B), and the thousand grain weight is increased.
Example 2 genetic analysis of Rice macromutant (dlz)
Hybridizing dlz mutant with wild Huanghua to obtain F 1 generation hybrid, planting F 2 segregation population with seeds of F 1 plant, collecting seeds after the seeds are ripe, completely drying, and measuring thousand seed weight. The average thousand kernel weight of wild type Huanghua is about 21 g, and the test result of the F 2 group shows that the large-kernel single plant occupies a small number in the group (figure 3). Because thousand grain weight is quantitative, the size of the thousand grain weight is continuously distributed in the population, 23 g is taken as a demarcation point, the thousand grain weight is greater than 23 g and is determined as large grain character, and the separation ratio of the normal character and the large grain character of two populations of F 2 -1 and F 2 -2 is in accordance with 3:1 (χ 2＝1.44<χ2 (0.05,df＝1) =3.84), which indicates that the large grain character of the mutant is generated by one recessive nuclear gene mutation.
EXAMPLE 3 cloning of Rice macromutant DLZ Gene
The gene cloning of the mutant adopts a SIMM method, namely, the mutant is hybridized with the original wild parent to construct F 2 generation population, and the gene positioning is carried out by resequencing. Specifically, dlz mutants are hybridized with wild Huanghua, 30 mutant plants with extreme phenotypes (more than 25 g thousand grains) are selected from F 2 population, genomic DNA of leaves is extracted respectively and mixed in equal quantity, library construction is carried out according to a sequencing library construction standard flow of an Illumina Hiseq2000 sequencing platform, and re-sequencing is carried out through PE 101. The sequencing data were analyzed by SIMM to locate the mutation site, and finally located at a 450kb interval at the end of chromosome 12 of rice, which includes four single base mutations, located in the coding regions of the LOC_Os12g41220, LOC_Os12g41820 and LOC_Os12g41910 genes, respectively, and two adjacent bases of the LOC_Os12g41820 gene were mutated (FIG. 4).
Another three F 1 individual plants of seed were taken to plant F 2 populations, each population was planted with about 600 to 800 individual plants, totaling 2100 plants. The genotype of the three mutation sites of each individual plant is identified by using an HRM method, 80 recombinant individual plants are screened from the genotype, and the grain width and thousand-grain weight of the recombinant individual plants and the non-recombinant individual plants are examined. As a result, it was found that the thousand kernel weight of the heterozygous strain was between that of the wild-type strain and that of the mutant strain in the non-recombinant strain, indicating that dlz gene had a semi-dominant effect: when both loc_os12g41820 and loc_os12g41910 are heterozygous, there is no difference in thousand kernel weight between the wild type and mutant of loc_os12g41220 sites; when both loci loc_os12g41220 and loc_os12g41820 are heterozygous, there is no difference in thousand kernel weight between wild type and mutant at locus loc_os12g 41910; the thousand seed weight difference between wild type and heterozygous, mutant at position 41820 of LOC_Os12g41820 alone was significant and the trend of the difference was the same as that of the non-recombinant individual (FIG. 5). The experiment result shows that LOC_Os12g41820 is DLZ gene.
In wild Huanghua (indica rice), the genome DNA of the DLZ gene has the total length of 6146bp and the sequence as shown in SEQ ID NO: 1. The gene has 9 exons, which are respectively shown in SEQ ID NO:1, from 831 to 1151, from 1502 to 1541, from 2298 to 2427, from 2569 to 3056, from 3148 to 3228, from 3364 to 3462, from 3643 to 3756, from 3840 to 3911, from 4498 to 4566. The full length of the coding region (CDS) of the gene is 1635bp, and the sequence is shown in SEQ ID NO:2, the CDS codes 1 protein containing 544 amino acids, and the amino acid sequence is shown in SEQ ID NO: 3. In the rice large grain mutant (DLZ) provided by the invention, two base mutations occur on the 4 th exon of the DLZ gene, specifically the nucleotide sequence of SEQ ID NO:2 from AAA to TTA (see SEQ ID NO: 4), resulting in the amino acid sequence of SEQ ID NO:3 from lysine (Lys) to leucine (Leu) (see SEQ ID NO: 5). Comparing japonica rice variety Nippon (typical japonica rice genome donor) with indica rice variety Huanghua, it was found that the nucleotide sequence of DLZ gene (see SEQ ID NO: 20) differs mainly in the non-coding region, and that the coding region has only one SNP site on the 6 th exon, specifically in SEQ ID NO:2, yellow Huazhan is G, japanese is A, and the SNP locus is located at the 1170 th base position of SEQ ID NO:3 (see SEQ ID NO: 21) but does not alter the encoded amino acid (see SEQ ID NO: 3), indicating that the DLZ gene differs less between japonica and indica.
EXAMPLE 4 analysis of expression pattern of DLZ Gene in Rice tissues and organs
Primers were designed based on the cDNA sequence of the DLZ gene, the upstream primer being 820qF:5'-AGTCCAGGCGTATACAGTGC-3' (SEQ ID NO: 6), the downstream primer is 820qR:5'-TCAGAGCAATCCTGACACCA-3' (SEQ ID NO: 7). Meanwhile, a primer is designed by taking the rice Ubiquitin gene as an internal reference, and the upstream primer is UBqF:5'-CAACCAGCTGAGGCCCAAGAA-3' (SEQ ID NO: 8), the downstream primer being UBqR:5'-CCAGGGAGATAACAACGGAAGC-3' (SEQ ID NO: 9). Extracting total RNA of tissues such as roots, stems, leaves, tillering buds, palea, pistils, anthers, young ears with different lengths, seeds 1 to 7 days after fertilization and the like of wild Huanghua, respectively, synthesizing cDNA templates, and analyzing the expression level of DLZ genes by adopting a real-time fluorescence quantitative PCR method. As a result, as shown in FIG. 6, the gene was expressed highly in each tissue and organ detected, and the difference in the expression level was small.
EXAMPLE 5 site-directed knockout of the DLZ Gene from indica and japonica
Target site sequence Target1 for 1 st CRISPR site-directed mutagenesis was selected on exon 1 of DLZ gene: 5'-CCTTCCTGGTCGACCGGCATTGG-3' (SEQ ID NO: 10), selecting on exon 3 the Target site sequence Target2 for site-directed mutagenesis of CRISPR 2:5'-GCTGTTCGTGTTGGATCGCTTGG-3' (SEQ ID NO: 11). Synthesis of the adapter primer U3-Target1-linkerF：5'-ggcACCTTCCTGGTCGACCGGCAT-3'(SEQ ID NO：12),U3-Target1-linkerR：5'-aaacATGCCGGTCGACCAGGAAGG-3'(SEQ ID NO：13),U6a-Target2-linkerF：5'-gccGCTGTTCGTGTTGGATCGCT-3'(SEQ ID NO：14),U6a-Target2-linkerR：5'-aaacAGCGATCCAACACGAACAG-3'(SEQ ID NO：15). with sticky ends two pairs of adapter primers were dissolved with ddH 2 O to 10. Mu.M working solution, 10. Mu.L of each of F and R primers was added to 80. Mu.L of ddH 2 O and mixed and diluted to 1. Mu.M, treated at 90℃for 30s, and cooled to room temperature to complete annealing. Then, T4DNA LIGASE is respectively connected into pYLsgRNA-U3 or pYLsgRNA-U6a plasmid which is tangential by BsaI enzyme to obtain trace U3:Target 1-gRNA and U6 a:Target 2-gRNA expression cassettes. And (3) taking the expression cassette as a template, performing two rounds of PCR amplification, adding BsaI enzyme cleavage sites, and connecting into PYLCRISPR/Cas9-MH (B) final vectors which are tangentially processed by BsaI enzyme to finally obtain the Pubi::: cas 9-U3::: target1-gRNA-U6 a::: target2-gRNA expression cassette. PCR templates and primer combinations are shown in Table 1, general primer sequences are shown in Table 2, and identification of positive clones is performed by bacterial liquid PCR detection using three pairs of primers, namely SP-L and U3-Target1-linker R, U3-Target1-linkerF and U6a-Target2-linker R, U6a-Target2-linkerF and SP-R, respectively, wherein the SP-L primer is used for sequencing of U3-Target1, and the SP-R is used for sequencing of U6a-Target 2.
Pubi:: cas9-U3:: target1-gRNA-U6 a::: target2-gRNA expression cassettes are respectively transferred into calli generated by the induction of flowers 11 and yellow Huazhan seeds in japonica rice varieties through an agrobacterium-mediated rice genetic transformation method, and a transgenic plant is obtained through hygromycin screening and differentiation regeneration of resistant calli. The genome DNA of the plant leaf is extracted by using a CTAB method, and primer pairs U3-Target1-linkerF and U6a-Target2-linkerR are used for positive detection and analysis of T-DNA insertion of transgenic plants, and the positive plants can amplify bands with about 600 bp. Genomic DNA from positive plants was re-used for primer pair 820-Target1-F:5'-CTGACATGGGCGCACATG-3' (SEQ ID NO: 16) and 820-Target2-R:5'-CCTCGTATCCTTGCAGCAACTT-3' (SEQ ID NO: 17) amplified an approximately 1.84kb DNA segment comprising two target sites, and reused primer pair 820-T1-SEQ:5'-GGTGATGCACACGAAGAAGC-3' (SEQ ID NO: 18) and 820-T2-SEQ:5'-TCCCTAGTTGCATCCGTTTG-3' (SEQ ID NO: 19) were used to sequence Target1 and Target2, respectively. As shown in FIG. 7A, the sequencing result shows that different base insertions or deletions occur at two target sites, so that the reading frame of the DLZ gene is changed, and both chromosomes are mutated, so that the generated positive plants are mutants with the function of the DLZ gene lost, the grain width of the positive plants is greatly increased compared with that of the wild type flower 11 or yellow Hua (FIG. 7B), and the fixed-point knockout of the DLZ genes of japonica rice and indica rice is proved to lead to the obvious increase of the seed size.
Example 6 dlz mutant Gene has remarkable yield-increasing Effect in heterozygous State
As shown in FIG. 8, the large grain mutant (dlz) has the advantages of larger grain width and increased thousand grain weight, the plant height is reduced compared with the wild Huanghua, but the stems are thicker, the spike number is reduced, the spike length is shortened, the glume number per spike is also obviously reduced, and the yield of the homozygous mutant is reduced by combining the characteristics. The plant height, the spike number, the spike length and the glume flower number of the large-grain mutant gene are similar to those of wild type Huanghua under the heterozygous state, no obvious difference exists, the grain width and the thousand grain weight of the heterozygous plant are higher than those of the wild type Huanghua and lower than those of the homozygous mutant, the stalk is stronger than that of the wild type material, the yield of the single plant is obviously increased than that of the wild type Huanghua, and the large-grain mutant gene has the potential of increasing the yield of rice under the heterozygous state and can be applied to the production of hybrid rice.
Example 7 inhibition of expression level of DLZ Gene Rice grain width was increased
Based on DLZ gene, primers were designed separately
RNAi-SpeI-1F：
5’-CACGTGGACCACTAGTATGTGGATGTATGGCTATTTCTGGA-3’(SEQ ID NO：22)，
RNAi-SpeI-1R：
5’-GTCCGTACCAACTAGTTCGTATCCTTGCAGCAACTTATTCA-3’(SEQ ID NO：23)，
RNAi-BamHI-2F：
5’-TGAATTCGCTGGATCCTCGTATCCTTGCAGCAACTTATTCA-3’(SEQ ID NO：24)，
RNAi-BamHI-2R：
5’-GTCGACTGGAGGATCCATGTGGATGTATGGCTATTTCTGGA-3’(SEQ ID NO：25)。
The cDNA of the DLZ gene is used as a template to amplify 214bp fragments respectively, and the fragments are respectively connected to the enzyme cutting sites SpeI and BamHI of the expression vector Ubi-intron In forward and reverse directions by an In-Fusion method to construct the RNAi vector. The vector is transferred into flower 11 in japonica rice by agrobacterium-mediated rice genetic transformation method, and the expression quantity analysis is carried out on the obtained T 0 generation transgenic plant. As shown in FIG. 9, the expression level of the endogenous DLZ gene of the transgenic positive plant is obviously inhibited, and the grain width is obviously increased, which indicates that the expression level of the DLZ gene can be increased, and the grain width is positively correlated with the expression level of the DLZ gene.
Example 8 Fine Regulation of the expression level of the DLZ Gene by promoter editing
8 CRISPR target sites are arranged in a 2087bp promoter region (SEQ ID NO: 26) upstream of an ATG (translation initiation site) of a DLZ gene, TP1：5'-TTTGACAGCTTCCTGATCTT-3'(SEQ ID NO：27),TP2：5'-CAAGTAAGATGCCAAGAATG-3'(SEQ ID NO：28),TP3：5'-TTGTCAACGGGAGAACAAC-3'(SEQ ID NO：29),TP4：5'-TAGGATATTTGAGCTACGG-3'(SEQ ID NO：30),TP5：5'-TAGAAAGAAGTCTGGAGCA-3'(SEQ ID NO：31),TP6：5'-AACGCCAGCTTGAGGGCAG-3'(SEQ ID NO：32),TP7：5'-TTCTCGTCGTTTCTTGCGTG-3'(SEQ ID NO：33),TP8：5'-GTGTGTGGGTTGACCGAAT-3'(SEQ ID NO：34). joint primers with sticky ends are respectively synthesized, a Pubi::Cas9-U3::TP3-gRNA-U3::TP6-gRNA-U6a::TP1-gRNA-U6a::TP2-gRNA-U6b::TP4-gRNA-U6b::TP5-gRNA-U6c::TP7-gRNA-U6c::TP8-gRNA expression cassette is constructed according to the method of example 5 as shown in table 3, a PCR template and a primer combination are shown in table 1, a general primer sequence is shown in table 2, and finally the expression cassette is transferred into flower 11 in japonica rice by an agrobacterium-mediated rice genetic transformation method. Primer 820Pro-2087bp-F:5'-GGAAAGGAAGAAAAGGCTAATATGCTCATC-3' (SEQ ID NO: 51) and 820Pro-2087bp-R:5'-ATGTCAGGATGTGCTTCTGGGACAC-3' (SEQ ID NO: 52) amplified the DLZ gene 2087bp promoter region of T 0 transgenic plants, and the electrophoresis result is shown in FIG. 10, and the amplified products of many transgenic plants are obviously smaller than 2087bp, which indicates that the DLZ gene promoter regions in the plants have fragment deletions with different degrees. The amplified product is connected into a pEASY-Blunt vector, and is sequenced by using M13F and M13R universal primers, and the sequencing result of the promoter of partial plants is shown as SEQ ID NO:53 (No. 8-5) SEQ ID NO:54 (No. 8-9 individuals). Wherein, the single plant 8-5 has about 750bp deletion between TP2 and TP 5; the large fragment of the individual No. 8-9 is deleted between TP2 and TP8, and the fragment of about 350bp between TP5 and TP6 is inserted upside down. These results indicate that promoter editing can produce abundant sequence variations.
Further, the change of the DLZ gene expression level of the transgenic plants was examined, and as a result, as shown in FIG. 10, the DLZ gene expression level of a few plants was suppressed to be equivalent to the effect of RNAi gene silencing (e.g., no. 8-22 individuals), and the DLZ gene expression level of a large part of plants was reduced by about half. The grain width phenotype of the transgenic plant is examined, and the plant with the grain width reduced by about half, the higher the inhibition degree of the DLZ gene expression level is, is found, the grain width is between the wild type plant and the gene knockout plant, namely, the grain width is equivalent to the phenotype of DLZ heterozygous mutant, and the fine regulation and control of the DLZ gene expression level can be realized through a promoter editing technology, so that the yield increasing effect is realized.
Example 9 homologous Gene analysis in different crops
A protein sequence encoded by the DLZ gene was input into NCBI database for BLASTP search to obtain homologous proteins predicted in crop genomes such as Brevibacterium reevesii (Brachypodium distachyon) (SEQ ID NO: 55), hordeum vulgare (Hordeum vulgare) (SEQ ID NO: 56), oryza sativa (Oryza brachyantha) (SEQ ID NO: 57), panicum mille (Panicum hallii) (SEQ ID NO: 58), setaria sativa (SETARIA ITALICA) (SEQ ID NO: 59), zea mays (SEQ ID NO: 60), sorghum (Sorgum bicolor) (SEQ ID NO: 61), etc. The amino acid sequences of the rice DLZ proteins and the homologous proteins are input into the ClustalW2 website (https:// www.ebi.ac.uk/Tools/msa/ClustalW /) for sequence alignment, and the results show that the homologous proteins from different plants all have very similar conserved sequences and have very high homology with each other (figure 11), which shows that the DLZ proteins play a very important role in function conservation in seed development of different plants.
The DLZ genes of the 7 crops are mutated by using CRISPR/Cas9 technology, and the obtained transgenic positive plants are observed in the shape of seed particles, so that the result shows that the transgenic positive plants are mutants with the function lost of the DLZ genes, the DLZ genes cannot be normally expressed, the seed particle width of the transgenic positive plants is greatly increased compared with the wild type of the corresponding crops, and the seed particle width of the transgenic positive plants is greatly increased, and the transgenic positive plants show the properties of large grain and spike, so that the homologous genes of DLZ of different species with the fixed point mutation cause the obvious increase of the seed size.
Table 1 CRISPR template primer combinations for amplifying gRNA expression cassettes in vector construction
Table 2 CRISPR general primer sequences for amplifying gRNA expression cassettes in vector construction
Table 3 target specific primer sequences for amplifying gRNA expression cassettes in promoter-edited CRISPR vector construction
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Sequence listing
<110> Shenzhen market crop molecular design Breeding institute
Front-end laboratory of crop design (Beijing) Co., ltd
<120> Gene regulating plant seed size and use thereof
<150> 201910051525.0
<151> 2019-01-21
<160> 61
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6146
<212> DNA
<213> Oryza sativa
<400> 1
cactgcccaa ttgcccatgc tccagacttc tttctactcc tacattccac atatctccat 60
ggacagtaac tcctcccaag ctaccacttc aaccctaatc ccctctctct ctcttccgca 120
gaggtagagt gagagagatg gtcagatagc tagattgata tccctctctc tctctcacag 180
acatctcttt ttgcaagatc tcttcttgtt catcatcttc ttcttttttt ctcccccttt 240
tgcttcacca atccatcttt tgtcacgaga tgtgaccgag ctgaagctag tagtagtgga 300
gcagcgaaag caagtacgcc aagaaaaaaa aaaggaagaa gaaagaagaa agaaagaaag 360
aaaaaaacgc cagcttgagg gcagagggca aaagcggcga cgaggagcag tggccaaagc 420
tcagattctt cccgtgggct atttttacca cccgcatccc ctctctttga gccccttggc 480
cgattcattc accgacgcaa agatccaacc cctcttcagg tgtcggcaga tgccgccttt 540
gtgaggtttc cagtgggggg atttctcgtc gtttcttgcg tgcggttgcg ttcttgatcc 600
agtgagcgca cggatatatc cgccctggtt tagtagagag agagagagag agagagagag 660
agagagagag agagagagag agagggggtt cttgattgag ttccaagtgt tggattgggt 720
tcttggagct gttggattgg gtttttttgg gagagagatg ggggtttgga ggtgtgtggg 780
ttgaccgaat tggatcaaga ttattgcggg aggggggggg gggggttgca atggcggatt 840
tggggctgtg gaagcaaggg tggaggtggg tggtgtccca gaagcacatc ctgacatggg 900
cgcacatggc ggcgagcggc ggcaccgaga ggctggcctt cctggtcgac cggcattggc 960
ccgccgtgtc ccgggcctgc gtgagctccg gccgcctcgc gctcgccgcg ctgcggcaat 1020
ggcgcggctg cgcggcgcgc gggatcctgg agatggctag cctgggccct gcgtccgtgt 1080
tcgtcatcct ctggagcttc ttcgtgtgca tcacctcgcc ggcgtgcgcc ctctacgcgc 1140
tcctgggcat ggtacggcat gcaagtcttg cttgctttgc gctttcgcct tgatgatgta 1200
gtggattatg gataacgatt tgtgcgcgtt ctaaatcttg tcatgtgctc gtctttcttt 1260
ttttcttctt tttatcaagg gtggattgca tgttaggtta cctttctttt cgaaaagtat 1320
agttaaagtg gtaattggtg gtacaaaagt agtatgtcat tacactttca tgagattgat 1380
cagtttgatg tgtttctaga ttcatttatg ctttagttat tgcaagttta tactacactt 1440
cagtaattca cacgtgctgt ttctagatgt tattttggaa ccgttcacag tattttaagc 1500
atcatttgca ttagaaagtt ttatctagtt tgtcttgcta gaggaaggag cacatggaaa 1560
ctaacacttg catatttagg gataagcact actggttcta ttcctatttt gtgtatgtta 1620
gctaatgtgt ttcttgctga gtggttcagt ttcaggttca ttagcagatt atcttattgg 1680
ttgatttatg tcaaaatact taaggtcaat tcgtagtttg cacagtgtac ttcaataaca 1740
tgaatgcaac tcgtttcttt gttcagcctg atatttatga aaaatcttat aatgtgatac 1800
tgtgtttaat atgtatgaac ctgtctagag aattactagc tagtgaaatt ctacttgttt 1860
catttcacac aaagtcaact atgggtagac tggttcatga ccatttattt aggctctggt 1920
acacctgtaa ctactgctgt agttgactat atgacttact tatgctggtt ttctactgtg 1980
gtatgagttt ctccctttgg gataccacct gtgttcagtg gaatgtcagg tagtatctga 2040
ccttttcagc tagattgcac tggataaatt atactgaaat aagcaatagg aatgaattcc 2100
aggactatgt ctcctcttgt tctctccttg caattcctct tattgcaatg cagactgaac 2160
cacttgtttt tactgtcatc tggcatactt gttcagttag taacttctac ttgcgagtaa 2220
ggatgcaaga tttcccatgt aagatggata ctataaatat cattttgtct aattgcttaa 2280
tacctttctt ttttcaggga gctgctgggg cagtcattca ttacatgggc tatacgcctg 2340
gtcttttcat tgtaggatta tttggaatat tgattatgtg gatgtatggc tatttctgga 2400
ttacaggaat gcttctgatt gctggaggtt tgttttatct taatatttaa gtctgttcat 2460
aatgataatt ttgtgttttt gtttgtcaaa tccataaatt tttcttcctc cctagttgca 2520
tccgtttgat tcttttgacc taaaggagga tcctctctgg taatgcaggc tgtatgtgct 2580
ctttgaaaca tgcacgattt gtgatacctg tgttggctat gtatgctgtt tattgtgtgg 2640
ctgttcgtgt tggatcgctt ggtgtcttct tgacattgaa tctttctttc ctgacaaatg 2700
atcttctgaa taagttgctg caaggatacg agggaagcac agaagaaaga cagtttgaag 2760
agccaaaaca ttctgatcct gtcatggatg agttctatcg cagttgtgaa tttccctctg 2820
ctcctgatag tgaacctgag actgtttctt ctgcaaagcc cttttgctca acacccgtcc 2880
aggatgtgtt gcatgtacag aaagaggcat ctcctagcaa agtagtgaaa tcggattctg 2940
tttcattgga tgagatgaag aggatcatgg atggtttgac ccattatgaa gttttgggta 3000
ttcctcggaa tagaagtatt gatcaaaaga ttctgaaaaa ggagtaccac agaatggtaa 3060
taaaccacgg ccttctatac aagggaaaat gagaaattca tgttacaatt acttcatttt 3120
catggtacgt atgctttatt tgtctaggtc ctgcttgtac atcctgataa aaatatggga 3180
aatccactgg cctgtgaatc attcaaaaag cttcagtcag cttatgaggt aaactacaat 3240
ggaagtttat gtcttttctc ttccttgatt atattacagt taaatctggt tgaatatctg 3300
ctcttgatac caaccatggc ttctatacct ggataaaggg taatcattgt agttatgctg 3360
caggtactct cagatttcac aaagaaaaac acttacgacg accaactgag gaaagaagaa 3420
tcacgtaaaa tgactcagag atcacgtgtt gtctctcaac aggtgggttc tagttttcac 3480
aaatttagaa tccacatggt tggattattt ctttaacata tcttatcaat tatccaagca 3540
tacgaatgca gtttattcat gctctcatgt ccttgaccta ctgacctact tgctgttttc 3600
ctttatgggg cccatttgta atttgataaa ctcatcttgc agactggggt agagtttctc 3660
tccgaagagt ccaggcgtat acagtgcaca aagtgtggta attttcatct gtggatatgt 3720
accaagaaaa gcaaagcaaa agcaagatgg tgtcaggttt ggaggccaga attttttttt 3780
caggtacttt taatcgagag tgttcttaca gctaattttg tgggaaccat gtactgtagg 3840
attgctctga ttttcatcca gctaaggatg gagatggatg ggtggaaaat aaattttcgt 3900
catccttcaa ggtaatgttt tataagcaca tcatatgaag agttcacttt attttactta 3960
atgcttgcct tctacagtac tcatagacag agatctagtg tcaatacaat tttaactact 4020
agaaaatgga aattgagtac atattgattt cgaacaaatg gagaatgagg ttttatgaat 4080
ggaagcacaa tgttctgaat gttttgatac aaaattaccg ggcgctgttt cccactgtca 4140
agcttcagtt cctagtactt gttattgcct gaagttagtc atgtgtgttc cgagaccaac 4200
tttggacttg agcaagctca gttttagctg tgtcaagctg atgatctttt atcttctaat 4260
tgtattccac ctaaagaaag catctcattc caagtgttag gtacagtcat tttgttcatt 4320
ccataagcaa cttattctga ctataaggtg agattcagaa attactcagc ttaaaaatgt 4380
gcacacattt tgtagtttcc aactataatg tgtaaattct tcacttctct ttattgaact 4440
ataatgtgta aattcttcag tactctttgt tgaatcaaag tgcattgttc acttcaggaa 4500
atacctcgag cttttgtttg tgcggagagt aaggtatttg atgtgtctga atgggctact 4560
tgccaggtga gtgtctgacg atgttttata tgtttgattt aagttgacat gtatgtgcat 4620
ttgcagcagt gatttttgga tgtctcaatt gatttgatgt catctccata tgcatatttt 4680
tatactcggt tctctgctgt tttcgatgtc ttaactgact atagatatgc ctttggtcaa 4740
ttgactttgt tcagttttgt atttgatgca tattcaaacg tccagattga ctgttttact 4800
ttaaaaattg tttcagttgg taaatgaaaa tttgcttact tcattggaga taggataatt 4860
catgcatgcc atagcccata gccttatttt tctgtgtcaa gtttgtcatg gctataataa 4920
acacaacata ttaatcgcac ccgcatgtca tcccgactga tatctcaatt attgacatac 4980
ctatctaaga gaagagccaa caatgatgaa agtaaaggac taatttggct gtgcaaaatt 5040
ggaccaaaag tttattttac atttcatact tgcttcattc aacataaaca tcaaaatctg 5100
gtacgccaat tttctggcga tacatacagg gtcttgtgat aggttcattt gcatacatta 5160
aaaatgggag cctttctaac tctgttttct ttgcttgatt gtctagggca tggagtgcaa 5220
acctaacact cacggcccat cttttatggt aaacatggtt ggcgcagata ggatgtctca 5280
gagatcctac agttctcgct atccctttag tttgaatgct gagatgatcc ctgaagatga 5340
atttgagcta tggcttcaac aagcattggc atcaggtgtc ttctctgaca gcccgaaacg 5400
caggaaaagc tggagcccct tcaaactacc tcaaaaaggg ataaaaagtt ggcggcgatc 5460
ctcataaggg catagcatta aacagcatgg atctcacctg agtacaacac tgaaaaaggc 5520
tatactcttt gtgaatgtaa atagactgac caacaatttg cctggatgag caactaattt 5580
tgtccaaaaa gagacactga aacaaggggg gtaaaaggaa caaacgctta agacatgact 5640
gcaatgaatc tgactgttga aattagtgtt ctctgcaatg agatcccgcg agttttatcc 5700
gaaaaggtca gatactggga tggcgtgtca ttcatcagtt catcctaaag ctcggaaggg 5760
tatctctgta gcatgttaac ttcagtagtt ttaggggatc ggcatctgag agaatttcaa 5820
aacttcatac ctggttgcca gcataagttc tgcaggtgtt gaaaagttgt tgatcagagt 5880
agcaatttaa ggtctgatgt ttctggggaa cagtaggaga gaaaaaaatg acaaaaaaaa 5940
gagagagttg gttgtaaata catgaaaagt tttcatcaga aattagtatt gtaacattgt 6000
acactgtgat tacatcctgt gcaatactcc cataattcag atctgtgttg taatacacta 6060
catacatcct acaattttct ggtgataata gagatctaat tctcacctat tatcgttatt 6120
tatggttagt cagttactgc tctgta 6146
<210> 2
<211> 1635
<212> DNA
<213> Oryza sativa
<400> 2
atggcggatt tggggctgtg gaagcaaggg tggaggtggg tggtgtccca gaagcacatc 60
ctgacatggg cgcacatggc ggcgagcggc ggcaccgaga ggctggcctt cctggtcgac 120
cggcattggc ccgccgtgtc ccgggcctgc gtgagctccg gccgcctcgc gctcgccgcg 180
ctgcggcaat ggcgcggctg cgcggcgcgc gggatcctgg agatggctag cctgggccct 240
gcgtccgtgt tcgtcatcct ctggagcttc ttcgtgtgca tcacctcgcc ggcgtgcgcc 300
ctctacgcgc tcctgggcat gggagctgct ggggcagtca ttcattacat gggctatacg 360
cctggtcttt tcattgtagg attatttgga atattgatta tgtggatgta tggctatttc 420
tggattacag gaatgcttct gattgctgga ggctgtatgt gctctttgaa acatgcacga 480
tttgtgatac ctgtgttggc tatgtatgct gtttattgtg tggctgttcg tgttggatcg 540
cttggtgtct tcttgacatt gaatctttct ttcctgacaa atgatcttct gaataagttg 600
ctgcaaggat acgagggaag cacagaagaa agacagtttg aagagccaaa acattctgat 660
cctgtcatgg atgagttcta tcgcagttgt gaatttccct ctgctcctga tagtgaacct 720
gagactgttt cttctgcaaa gcccttttgc tcaacacccg tccaggatgt gttgcatgta 780
cagaaagagg catctcctag caaagtagtg aaatcggatt ctgtttcatt ggatgagatg 840
aagaggatca tggatggttt gacccattat gaagttttgg gtattcctcg gaatagaagt 900
attgatcaaa agattctgaa aaaggagtac cacagaatgg tcctgcttgt acatcctgat 960
aaaaatatgg gaaatccact ggcctgtgaa tcattcaaaa agcttcagtc agcttatgag 1020
gtactctcag atttcacaaa gaaaaacact tacgacgacc aactgaggaa agaagaatca 1080
cgtaaaatga ctcagagatc acgtgttgtc tctcaacaga ctggggtaga gtttctctcc 1140
gaagagtcca ggcgtataca gtgcacaaag tgtggtaatt ttcatctgtg gatatgtacc 1200
aagaaaagca aagcaaaagc aagatggtgt caggattgct ctgattttca tccagctaag 1260
gatggagatg gatgggtgga aaataaattt tcgtcatcct tcaaggaaat acctcgagct 1320
tttgtttgtg cggagagtaa ggtatttgat gtgtctgaat gggctacttg ccagggcatg 1380
gagtgcaaac ctaacactca cggcccatct tttatggtaa acatggttgg cgcagatagg 1440
atgtctcaga gatcctacag ttctcgctat ccctttagtt tgaatgctga gatgatccct 1500
gaagatgaat ttgagctatg gcttcaacaa gcattggcat caggtgtctt ctctgacagc 1560
ccgaaacgca ggaaaagctg gagccccttc aaactacctc aaaaagggat aaaaagttgg 1620
cggcgatcct cataa 1635
<210> 3
<211> 544
<212> PRT
<213> Oryza sativa
<400> 3
Met Ala Asp Leu Gly Leu Trp Lys Gln Gly Trp Arg Trp Val Val Ser
1 5 10 15
Gln Lys His Ile Leu Thr Trp Ala His Met Ala Ala Ser Gly Gly Thr
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Val Ser Ser Gly Arg Leu Ala Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Ala Ala Arg Gly Ile Leu Glu Met Ala Ser Leu Gly Pro
65 70 75 80
Ala Ser Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Ile Thr Ser
85 90 95
Pro Ala Cys Ala Leu Tyr Ala Leu Leu Gly Met Gly Ala Ala Gly Ala
100 105 110
Val Ile His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Ile Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Phe Val Ile Pro Val Leu Ala Met Tyr Ala Val Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Ser Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Arg Gln Phe Glu Glu Pro Lys His Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Phe Pro Ser Ala Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Phe Cys Ser Thr Pro Val Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Ala Ser Pro Ser Lys Val Val Lys Ser
260 265 270
Asp Ser Val Ser Leu Asp Glu Met Lys Arg Ile Met Asp Gly Leu Thr
275 280 285
His Tyr Glu Val Leu Gly Ile Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Ile Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Thr Tyr Asp
340 345 350
Asp Gln Leu Arg Lys Glu Glu Ser Arg Lys Met Thr Gln Arg Ser Arg
355 360 365
Val Val Ser Gln Gln Thr Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Leu Trp Ile Cys Thr
385 390 395 400
Lys Lys Ser Lys Ala Lys Ala Arg Trp Cys Gln Asp Cys Ser Asp Phe
405 410 415
His Pro Ala Lys Asp Gly Asp Gly Trp Val Glu Asn Lys Phe Ser Ser
420 425 430
Ser Phe Lys Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys Val
435 440 445
Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys Pro
450 455 460
Asn Thr His Gly Pro Ser Phe Met Val Asn Met Val Gly Ala Asp Arg
465 470 475 480
Met Ser Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asn Ala
485 490 495
Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala Leu
500 505 510
Ala Ser Gly Val Phe Ser Asp Ser Pro Lys Arg Arg Lys Ser Trp Ser
515 520 525
Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser Ser
530 535 540
<210> 4
<211> 6146
<212> DNA
<213> Oryza sativa
<400> 4
cactgcccaa ttgcccatgc tccagacttc tttctactcc tacattccac atatctccat 60
ggacagtaac tcctcccaag ctaccacttc aaccctaatc ccctctctct ctcttccgca 120
gaggtagagt gagagagatg gtcagatagc tagattgata tccctctctc tctctcacag 180
acatctcttt ttgcaagatc tcttcttgtt catcatcttc ttcttttttt ctcccccttt 240
tgcttcacca atccatcttt tgtcacgaga tgtgaccgag ctgaagctag tagtagtgga 300
gcagcgaaag caagtacgcc aagaaaaaaa aaaggaagaa gaaagaagaa agaaagaaag 360
aaaaaaacgc cagcttgagg gcagagggca aaagcggcga cgaggagcag tggccaaagc 420
tcagattctt cccgtgggct atttttacca cccgcatccc ctctctttga gccccttggc 480
cgattcattc accgacgcaa agatccaacc cctcttcagg tgtcggcaga tgccgccttt 540
gtgaggtttc cagtgggggg atttctcgtc gtttcttgcg tgcggttgcg ttcttgatcc 600
agtgagcgca cggatatatc cgccctggtt tagtagagag agagagagag agagagagag 660
agagagagag agagagagag agagggggtt cttgattgag ttccaagtgt tggattgggt 720
tcttggagct gttggattgg gtttttttgg gagagagatg ggggtttgga ggtgtgtggg 780
ttgaccgaat tggatcaaga ttattgcggg aggggggggg gggggttgca atggcggatt 840
tggggctgtg gaagcaaggg tggaggtggg tggtgtccca gaagcacatc ctgacatggg 900
cgcacatggc ggcgagcggc ggcaccgaga ggctggcctt cctggtcgac cggcattggc 960
ccgccgtgtc ccgggcctgc gtgagctccg gccgcctcgc gctcgccgcg ctgcggcaat 1020
ggcgcggctg cgcggcgcgc gggatcctgg agatggctag cctgggccct gcgtccgtgt 1080
tcgtcatcct ctggagcttc ttcgtgtgca tcacctcgcc ggcgtgcgcc ctctacgcgc 1140
tcctgggcat ggtacggcat gcaagtcttg cttgctttgc gctttcgcct tgatgatgta 1200
gtggattatg gataacgatt tgtgcgcgtt ctaaatcttg tcatgtgctc gtctttcttt 1260
ttttcttctt tttatcaagg gtggattgca tgttaggtta cctttctttt cgaaaagtat 1320
agttaaagtg gtaattggtg gtacaaaagt agtatgtcat tacactttca tgagattgat 1380
cagtttgatg tgtttctaga ttcatttatg ctttagttat tgcaagttta tactacactt 1440
cagtaattca cacgtgctgt ttctagatgt tattttggaa ccgttcacag tattttaagc 1500
atcatttgca ttagaaagtt ttatctagtt tgtcttgcta gaggaaggag cacatggaaa 1560
ctaacacttg catatttagg gataagcact actggttcta ttcctatttt gtgtatgtta 1620
gctaatgtgt ttcttgctga gtggttcagt ttcaggttca ttagcagatt atcttattgg 1680
ttgatttatg tcaaaatact taaggtcaat tcgtagtttg cacagtgtac ttcaataaca 1740
tgaatgcaac tcgtttcttt gttcagcctg atatttatga aaaatcttat aatgtgatac 1800
tgtgtttaat atgtatgaac ctgtctagag aattactagc tagtgaaatt ctacttgttt 1860
catttcacac aaagtcaact atgggtagac tggttcatga ccatttattt aggctctggt 1920
acacctgtaa ctactgctgt agttgactat atgacttact tatgctggtt ttctactgtg 1980
gtatgagttt ctccctttgg gataccacct gtgttcagtg gaatgtcagg tagtatctga 2040
ccttttcagc tagattgcac tggataaatt atactgaaat aagcaatagg aatgaattcc 2100
aggactatgt ctcctcttgt tctctccttg caattcctct tattgcaatg cagactgaac 2160
cacttgtttt tactgtcatc tggcatactt gttcagttag taacttctac ttgcgagtaa 2220
ggatgcaaga tttcccatgt aagatggata ctataaatat cattttgtct aattgcttaa 2280
tacctttctt ttttcaggga gctgctgggg cagtcattca ttacatgggc tatacgcctg 2340
gtcttttcat tgtaggatta tttggaatat tgattatgtg gatgtatggc tatttctgga 2400
ttacaggaat gcttctgatt gctggaggtt tgttttatct taatatttaa gtctgttcat 2460
aatgataatt ttgtgttttt gtttgtcaaa tccataaatt tttcttcctc cctagttgca 2520
tccgtttgat tcttttgacc taaaggagga tcctctctgg taatgcaggc tgtatgtgct 2580
ctttgaaaca tgcacgattt gtgatacctg tgttggctat gtatgctgtt tattgtgtgg 2640
ctgttcgtgt tggatcgctt ggtgtcttct tgacattgaa tctttctttc ctgacaaatg 2700
atcttctgaa taagttgctg caaggatacg agggaagcac agaagaaaga cagtttgaag 2760
agccaaaaca ttctgatcct gtcatggatg agttctatcg cagttgtgaa tttccctctg 2820
ctcctgatag tgaacctgag actgtttctt ctgcaaagcc cttttgctca acacccgtcc 2880
aggatgtgtt gcatgtacag aaagaggcat ctcctagcaa agtagtgaaa tcggattctg 2940
tttcattgga tgagatgaag aggatcatgg atggtttgac ccattatgaa gttttgggta 3000
ttcctcggaa tagaagtatt gatcaaaaga ttctgaaaaa ggagtaccac agaatggtaa 3060
taaaccacgg ccttctatac aagggaaaat gagaaattca tgttacaatt acttcatttt 3120
catggtacgt atgctttatt tgtctaggtc ctgcttgtac atcctgattt aaatatggga 3180
aatccactgg cctgtgaatc attcaaaaag cttcagtcag cttatgaggt aaactacaat 3240
ggaagtttat gtcttttctc ttccttgatt atattacagt taaatctggt tgaatatctg 3300
ctcttgatac caaccatggc ttctatacct ggataaaggg taatcattgt agttatgctg 3360
caggtactct cagatttcac aaagaaaaac acttacgacg accaactgag gaaagaagaa 3420
tcacgtaaaa tgactcagag atcacgtgtt gtctctcaac aggtgggttc tagttttcac 3480
aaatttagaa tccacatggt tggattattt ctttaacata tcttatcaat tatccaagca 3540
tacgaatgca gtttattcat gctctcatgt ccttgaccta ctgacctact tgctgttttc 3600
ctttatgggg cccatttgta atttgataaa ctcatcttgc agactggggt agagtttctc 3660
tccgaagagt ccaggcgtat acagtgcaca aagtgtggta attttcatct gtggatatgt 3720
accaagaaaa gcaaagcaaa agcaagatgg tgtcaggttt ggaggccaga attttttttt 3780
caggtacttt taatcgagag tgttcttaca gctaattttg tgggaaccat gtactgtagg 3840
attgctctga ttttcatcca gctaaggatg gagatggatg ggtggaaaat aaattttcgt 3900
catccttcaa ggtaatgttt tataagcaca tcatatgaag agttcacttt attttactta 3960
atgcttgcct tctacagtac tcatagacag agatctagtg tcaatacaat tttaactact 4020
agaaaatgga aattgagtac atattgattt cgaacaaatg gagaatgagg ttttatgaat 4080
ggaagcacaa tgttctgaat gttttgatac aaaattaccg ggcgctgttt cccactgtca 4140
agcttcagtt cctagtactt gttattgcct gaagttagtc atgtgtgttc cgagaccaac 4200
tttggacttg agcaagctca gttttagctg tgtcaagctg atgatctttt atcttctaat 4260
tgtattccac ctaaagaaag catctcattc caagtgttag gtacagtcat tttgttcatt 4320
ccataagcaa cttattctga ctataaggtg agattcagaa attactcagc ttaaaaatgt 4380
gcacacattt tgtagtttcc aactataatg tgtaaattct tcacttctct ttattgaact 4440
ataatgtgta aattcttcag tactctttgt tgaatcaaag tgcattgttc acttcaggaa 4500
atacctcgag cttttgtttg tgcggagagt aaggtatttg atgtgtctga atgggctact 4560
tgccaggtga gtgtctgacg atgttttata tgtttgattt aagttgacat gtatgtgcat 4620
ttgcagcagt gatttttgga tgtctcaatt gatttgatgt catctccata tgcatatttt 4680
tatactcggt tctctgctgt tttcgatgtc ttaactgact atagatatgc ctttggtcaa 4740
ttgactttgt tcagttttgt atttgatgca tattcaaacg tccagattga ctgttttact 4800
ttaaaaattg tttcagttgg taaatgaaaa tttgcttact tcattggaga taggataatt 4860
catgcatgcc atagcccata gccttatttt tctgtgtcaa gtttgtcatg gctataataa 4920
acacaacata ttaatcgcac ccgcatgtca tcccgactga tatctcaatt attgacatac 4980
ctatctaaga gaagagccaa caatgatgaa agtaaaggac taatttggct gtgcaaaatt 5040
ggaccaaaag tttattttac atttcatact tgcttcattc aacataaaca tcaaaatctg 5100
gtacgccaat tttctggcga tacatacagg gtcttgtgat aggttcattt gcatacatta 5160
aaaatgggag cctttctaac tctgttttct ttgcttgatt gtctagggca tggagtgcaa 5220
acctaacact cacggcccat cttttatggt aaacatggtt ggcgcagata ggatgtctca 5280
gagatcctac agttctcgct atccctttag tttgaatgct gagatgatcc ctgaagatga 5340
atttgagcta tggcttcaac aagcattggc atcaggtgtc ttctctgaca gcccgaaacg 5400
caggaaaagc tggagcccct tcaaactacc tcaaaaaggg ataaaaagtt ggcggcgatc 5460
ctcataaggg catagcatta aacagcatgg atctcacctg agtacaacac tgaaaaaggc 5520
tatactcttt gtgaatgtaa atagactgac caacaatttg cctggatgag caactaattt 5580
tgtccaaaaa gagacactga aacaaggggg gtaaaaggaa caaacgctta agacatgact 5640
gcaatgaatc tgactgttga aattagtgtt ctctgcaatg agatcccgcg agttttatcc 5700
gaaaaggtca gatactggga tggcgtgtca ttcatcagtt catcctaaag ctcggaaggg 5760
tatctctgta gcatgttaac ttcagtagtt ttaggggatc ggcatctgag agaatttcaa 5820
aacttcatac ctggttgcca gcataagttc tgcaggtgtt gaaaagttgt tgatcagagt 5880
agcaatttaa ggtctgatgt ttctggggaa cagtaggaga gaaaaaaatg acaaaaaaaa 5940
gagagagttg gttgtaaata catgaaaagt tttcatcaga aattagtatt gtaacattgt 6000
acactgtgat tacatcctgt gcaatactcc cataattcag atctgtgttg taatacacta 6060
catacatcct acaattttct ggtgataata gagatctaat tctcacctat tatcgttatt 6120
tatggttagt cagttactgc tctgta 6146
<210> 5
<211> 544
<212> PRT
<213> Oryza sativa
<400> 5
Met Ala Asp Leu Gly Leu Trp Lys Gln Gly Trp Arg Trp Val Val Ser
1 5 10 15
Gln Lys His Ile Leu Thr Trp Ala His Met Ala Ala Ser Gly Gly Thr
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Val Ser Ser Gly Arg Leu Ala Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Ala Ala Arg Gly Ile Leu Glu Met Ala Ser Leu Gly Pro
65 70 75 80
Ala Ser Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Ile Thr Ser
85 90 95
Pro Ala Cys Ala Leu Tyr Ala Leu Leu Gly Met Gly Ala Ala Gly Ala
100 105 110
Val Ile His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Ile Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Phe Val Ile Pro Val Leu Ala Met Tyr Ala Val Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Ser Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Arg Gln Phe Glu Glu Pro Lys His Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Phe Pro Ser Ala Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Phe Cys Ser Thr Pro Val Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Ala Ser Pro Ser Lys Val Val Lys Ser
260 265 270
Asp Ser Val Ser Leu Asp Glu Met Lys Arg Ile Met Asp Gly Leu Thr
275 280 285
His Tyr Glu Val Leu Gly Ile Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Ile Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Leu Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Thr Tyr Asp
340 345 350
Asp Gln Leu Arg Lys Glu Glu Ser Arg Lys Met Thr Gln Arg Ser Arg
355 360 365
Val Val Ser Gln Gln Thr Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Leu Trp Ile Cys Thr
385 390 395 400
Lys Lys Ser Lys Ala Lys Ala Arg Trp Cys Gln Asp Cys Ser Asp Phe
405 410 415
His Pro Ala Lys Asp Gly Asp Gly Trp Val Glu Asn Lys Phe Ser Ser
420 425 430
Ser Phe Lys Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys Val
435 440 445
Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys Pro
450 455 460
Asn Thr His Gly Pro Ser Phe Met Val Asn Met Val Gly Ala Asp Arg
465 470 475 480
Met Ser Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asn Ala
485 490 495
Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala Leu
500 505 510
Ala Ser Gly Val Phe Ser Asp Ser Pro Lys Arg Arg Lys Ser Trp Ser
515 520 525
Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser Ser
530 535 540
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 6
agtccaggcg tatacagtgc 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 7
tcagagcaat cctgacacca 20
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 8
caaccagctg aggcccaaga a 21
<210> 9
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 9
ccagggagat aacaacggaa gc 22
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 10
ccttcctggt cgaccggcat tgg 23
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 11
gctgttcgtg ttggatcgct tgg 23
<210> 12
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 12
ggcaccttcc tggtcgaccg gcat 24
<210> 13
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 13
aaacatgccg gtcgaccagg aagg 24
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 14
gccgctgttc gtgttggatc gct 23
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 15
aaacagcgat ccaacacgaa cag 23
<210> 16
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 16
ctgacatggg cgcacatg 18
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 17
cctcgtatcc ttgcagcaac tt 22
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 18
ggtgatgcac acgaagaagc 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 19
tccctagttg catccgtttg 20
<210> 20
<211> 6118
<212> DNA
<213> Oryza sativa
<400> 20
cactgcccaa ttgcccatgc tccagacttc tttctactcc tacattccac atatctccat 60
ggacagtaac tcctcccaag ctaccacttc aaccctaatc ccctctctct ctcttccgca 120
gaggtagagt gagagagatg gtcagatagc tagattgata tccctctctc tctctcacac 180
acatctcttt ttgcaagatc tcttcttgtt catcatcttc ttcttttttt ctcccccttt 240
tgcttcacca atccatcttt tgtcacgaga tgtggccgag ctgaagctag tagtagtgga 300
gcagcgaaag caagtacgcc aagaaaaaaa aaaggaagaa gaaagaagaa agaaagaaag 360
aaaaaaacgc cagcttgagg gcagagggca aaagcggcga cgaggagcag tggccaaagc 420
tcagattctt cccgtgggct atttttacca cccgcatccc ctctctttga gccccttggc 480
cgattcattc accgacgcaa agatccaacc cctcttcagg tgtcggcaga tgccgccttt 540
gtgaggtttc cagtgggggg atttctcgtc gtttcttgcg tgcggttgcg ttcttgatcc 600
agtgagcgca cggatatatc cgccctggtt tagtagagag agagagagag agagagagag 660
agagagagag aggttcttga ttgagttcca agtgttggat tgggttcttg gagctgttgg 720
attgggtttt tttgggagag agatgggggt ttggaggtgt gtgggttgac cgaattggat 780
caagattatt gcgggagggg gggggggggg ttgcaatggc ggatttgggg ctgtggaagc 840
aagggtggag gtgggtggtg tcccagaagc acatcctgac atgggcgcac atggcggcga 900
gcggcggcac cgagaggctg gccttcctgg tcgaccggca ttggcccgcc gtgtcccggg 960
cctgcgtgag ctccggccgc ctcgcgctcg ccgcgctgcg gcaatggcgc ggctgcgcgg 1020
cgcgcgggat cctggagatg gctagcctgg gccctgcgtc cgtgttcgtc atcctctgga 1080
gcttcttcgt gtgcatcacc tcgccggcgt gcgccctcta cgcgctcctg ggcatggtac 1140
ggcatgcaag tcttgcttgc tttgcgcttt cgccttgatg atgtagtgga ttatggataa 1200
cgatttgtgc gcgttctaaa tcttgtcatg tgctcgtctt tctttttttc ttctttttat 1260
caagggtgga ttgcatgtta ggttaccttt cttttcgaaa agtatagtta aagtggtaat 1320
tggtggtaca aaagtagtat gtcattacac tttcatgaga ttgatcagtt tgatgtgttt 1380
ctagattcat ttatgcttta gttattgcaa gtttatacta cacttcagta attcacacgt 1440
gctgtttcta gatgttattt tggaaccgtt cacagtattt taagcagaga aagttttatc 1500
tagtttgtct tgctagagga aggagcacat ggaaactaac acttgcatat ttagggataa 1560
gcactactgg ttctattcct attttgtgta tgttagctaa tgtgtttctt gctgagtggt 1620
tcagtttcag gttcattagc agattatctt attggttgat ttatgtcaaa atacttaagg 1680
tcaattcgta gtttgcacag tgtacttcaa taacatgaat gcaactcgtt tctttgttca 1740
gcctgatatt tatgaaaaat cttataatgt gatactgtgt ttaatatgta tgaacctgtc 1800
tagagaatta ctagctagtg aaattctact tgtttcattt cacacaaagt caactatggg 1860
tagactggtt catgaccatt tatttaggct ctggtacacc tgtaactact gctgtagttg 1920
actatatgac ttacttctgc tggttttcta ctgtggtatg agtttctccc tttgggatac 1980
cacctgtgtt cagtggaatg tcaggtagta tctgaccttt tcagctagat tgcactggat 2040
aaattatact gaaataagca ataggaatga attccaggac tatgtctcct cttgttctct 2100
ccttgcaatt cctcttattg caatgcagac tgaaccactt gtttttacta tcatctggca 2160
tacttgttca gttagtaact tctacttgcg agtaaggatg caagatttcc catgtaagat 2220
ggatactata aatatcattt tgtctaattg cttaatacct ttcttttttc agggagctgc 2280
tggggcagtc attcattaca tgggctatac gcctggtctt ttcattgtag gattatttgg 2340
aatattgatt atgtggatgt atggctattt ctggattaca ggaatgcttc tgattgctgg 2400
aggtttgttt tatcttaata tttaagtctg ttcataatga taattttgtg tttttgtttg 2460
tcaaatccat aaatttttct tcctccctag ttgcatccgt ttgattcttt tgacctaaag 2520
gaggatcctc tctggtaatg caggctgtat gtgctctttg aaacatgcac gatttgtgat 2580
acctgtgttg gctatgtatg ctgtttattg tgtggctgtt cgtgttggat cgcttggtgt 2640
cttcttgaca ttgaatcttt ctttcctgac aaatgatctt ctgaataagt tgctgcaagg 2700
atacgaggga agcacagaag aaagacagtt tgaagagcca aaacattctg atcctgtcat 2760
ggatgagttc tatcgcagtt gtgaatttcc ctctgctcct gatagtgaac ctgagactgt 2820
ttcttctgca aagccctttt gctcaacacc cgtccaggat gtgttgcatg tacagaaaga 2880
ggcatctcct agcaaagtag tgaaatcgga ttctgtttca ttggatgaga tgaagaggat 2940
catggatggt ttgacccatt atgaagtttt gggtattcct cggaatagaa gtattgatca 3000
aaagattctg aaaaaggagt accacagaat ggtaataaac cacggccttc tatacaaggg 3060
aaaatgagaa attcatgtta caattacttc attttcatgg tacgtatgct ttatttgtct 3120
aggtcctgct tgtacatcct gataaaaata tgggaaatcc actggcctgt gaatcattca 3180
aaaagcttca gtcagcttat gaggtaaact acaatggaag tttatgtctt ttctcttcct 3240
tgattatatt acagttaaat ctggttgaat atctgctctt gataccaacc atggcttcta 3300
tacctggata aagggtaatc attgtagtta tgctgcaggt actctcagat ttcacaaaga 3360
aaaacactta cgacgaccaa ctgaggaaag aagaatcacg taaaatgact cagagatcac 3420
gtgttgtctc tcaacaggtg ggttctagtt ttcacaaatt tagaatccac atggttggat 3480
tatttcttta acatatctta tcaattatcc aagcatacga atgcagttta ttcatgctct 3540
catgtccttg acctactgac ctacttgctg ttttccttta tggggcccat ttgtaatttg 3600
ataaactcat cttgcagact ggggtagagt ttctctccga agagtccagg cgtatacagt 3660
gcacaaaatg tggtaatttt catctgtgga tatgtaccaa gaaaagcaaa gcaaaagcaa 3720
gatggtgtca ggtttggagg ccagaatttt tttttcaggt acttttaatc gagagtgttc 3780
ttacagctaa ttttgtggga accatgtact gtaggattgc tctgattttc atccagctaa 3840
ggatggagat ggatgggtgg aaaataaatt ttcgtcatcc ttcaaggtaa tgttttataa 3900
gcacatcata tgaagagttc actttatttt acttaatgct tgccttctac agtactcata 3960
gacagagatc tagtgtcaat agaattttaa ctactagaaa atggaaattg agtacatgtt 4020
gatttcgaac aaatggagaa tgaggtttta tgaatggaag cacaatgttc tgaatgtttt 4080
gatacaaaat taccgggcgc tgtttcccac tgtcaagctt cagttcctag tacttgttat 4140
tgcctgaagt tagtcatgtg tgttccgaga ccaactttgg acttgagcaa gctcagtttt 4200
agctgtgtca agctgatgat cttttatctt ctaattgtat tccacctaaa gaaagcatct 4260
cattccaagt gttaggtaca gtcattttgt tcattccata agcaacttat tctgactata 4320
gggtgagatt cagaaattac tcagcttaaa aatgtgcaca cattttgtag tttccaacta 4380
taatgtgtaa attcttcact tctctttatt gaactataat gtgtaaattc ttcagtactc 4440
tttgttgaat caaagtgcat tgttcacttc aggaaatacc tcgagctttt gtttgtgcgg 4500
agagtaaggt atttgatgtg tctgaatggg ctacttgcca ggtgagtgtc tgacgatgtt 4560
ttatatgttt gatttaagtt gacatgtatg tgcatttgca gcagtgattt ttggatgtct 4620
caattgattt gatgtcatct ccatatgcat atttttatac tcggttctct gctgttttcg 4680
atgtcttaac tgactataga tatgcctttg gtcaattgac tttgttcagt tttgtatttg 4740
atgcatattc aaacgtccag attgactgtt ttactttaaa aattgtttca gttggtaaat 4800
gaaaatttgc ttacttcatt ggagatagga taattcatgc atgccatagc ccatagcctt 4860
atttttctgt gtcaagtttg tcatggctat aataaacaca acatattaat tgcacctgca 4920
tgtcatcccg actgatatct caattattga catacctatc taagagaaga gccaacaatg 4980
atgaaagtaa aggactaatt tggctgtgca aaattggacc ataagtttat tttacatttc 5040
atacttgctt cattcaacat aaacatcaaa atctggtacg ccaattttct ggcgatacat 5100
acagggtctt gtgataggtt catttgcata cattaaaaat gggagccttt ctaactctgt 5160
tttctttgct tgattgtcta gggcatggag tgcaaaccta acactcacgg cccatctttt 5220
atggtaaaca tggttggcgc agataggatg tctcagagat cctacagttc tcgctatccc 5280
tttagtttga atgctgagat gatccctgaa gatgaatttg agctatggct tcaacaagca 5340
ttggcatcag gtgtcttctc tgacagcccg aaacgcagga aaagctggag ccccttcaaa 5400
ctacctcaaa aagggataaa aagttggcgg cgatcctcat aagggcatag cattgaacag 5460
catggatctc acctgagtac aacactgaaa aaggctatac tctttgtgaa tgtaaataga 5520
ctgaccaaca atttgcctgg atgagcaact aattttgtcc aaaaagagac tgaaacaagg 5580
ggggtaaaag gaacaaacgc ttaagacatg actgcaataa atctgactgt tgaaattagt 5640
gttctctgca atgagatccc gcgagtttta tccgaaaagg tcagacactg ggatggtgtg 5700
tcattcatca gttcatccta agctcggaag ggtatctctg tagcatgtta acttcagtag 5760
ttttagggga tcggcatctg agagaatttc aaaacttcat acctggttgc cagcataagt 5820
tctgcaggtg ttgaaaagtt gttgatcaga gtagcaattt aaggtctgat gtttctgggg 5880
aacagtagga gagaaaaaaa tgacaaaaaa aagagagagt aggttgtaaa tacatgaaaa 5940
gttttcatca gaaattagtg ttgtaacatt gtacactgtg attacatcct gtgcaatact 6000
cccataattc agatctgtgt tgtaatacac tacatacatc ctacaatttt ctggtgataa 6060
tagagatcta attctcacct attatcgtta tttatggtta gtcagttact gctctgta 6118
<210> 21
<211> 1635
<212> DNA
<213> Oryza sativa
<400> 21
atggcggatt tggggctgtg gaagcaaggg tggaggtggg tggtgtccca gaagcacatc 60
ctgacatggg cgcacatggc ggcgagcggc ggcaccgaga ggctggcctt cctggtcgac 120
cggcattggc ccgccgtgtc ccgggcctgc gtgagctccg gccgcctcgc gctcgccgcg 180
ctgcggcaat ggcgcggctg cgcggcgcgc gggatcctgg agatggctag cctgggccct 240
gcgtccgtgt tcgtcatcct ctggagcttc ttcgtgtgca tcacctcgcc ggcgtgcgcc 300
ctctacgcgc tcctgggcat gggagctgct ggggcagtca ttcattacat gggctatacg 360
cctggtcttt tcattgtagg attatttgga atattgatta tgtggatgta tggctatttc 420
tggattacag gaatgcttct gattgctgga ggctgtatgt gctctttgaa acatgcacga 480
tttgtgatac ctgtgttggc tatgtatgct gtttattgtg tggctgttcg tgttggatcg 540
cttggtgtct tcttgacatt gaatctttct ttcctgacaa atgatcttct gaataagttg 600
ctgcaaggat acgagggaag cacagaagaa agacagtttg aagagccaaa acattctgat 660
cctgtcatgg atgagttcta tcgcagttgt gaatttccct ctgctcctga tagtgaacct 720
gagactgttt cttctgcaaa gcccttttgc tcaacacccg tccaggatgt gttgcatgta 780
cagaaagagg catctcctag caaagtagtg aaatcggatt ctgtttcatt ggatgagatg 840
aagaggatca tggatggttt gacccattat gaagttttgg gtattcctcg gaatagaagt 900
attgatcaaa agattctgaa aaaggagtac cacagaatgg tcctgcttgt acatcctgat 960
aaaaatatgg gaaatccact ggcctgtgaa tcattcaaaa agcttcagtc agcttatgag 1020
gtactctcag atttcacaaa gaaaaacact tacgacgacc aactgaggaa agaagaatca 1080
cgtaaaatga ctcagagatc acgtgttgtc tctcaacaga ctggggtaga gtttctctcc 1140
gaagagtcca ggcgtataca gtgcacaaaa tgtggtaatt ttcatctgtg gatatgtacc 1200
aagaaaagca aagcaaaagc aagatggtgt caggattgct ctgattttca tccagctaag 1260
gatggagatg gatgggtgga aaataaattt tcgtcatcct tcaaggaaat acctcgagct 1320
tttgtttgtg cggagagtaa ggtatttgat gtgtctgaat gggctacttg ccagggcatg 1380
gagtgcaaac ctaacactca cggcccatct tttatggtaa acatggttgg cgcagatagg 1440
atgtctcaga gatcctacag ttctcgctat ccctttagtt tgaatgctga gatgatccct 1500
gaagatgaat ttgagctatg gcttcaacaa gcattggcat caggtgtctt ctctgacagc 1560
ccgaaacgca ggaaaagctg gagccccttc aaactacctc aaaaagggat aaaaagttgg 1620
cggcgatcct cataa 1635
<210> 22
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 22
cacgtggacc actagtatgt ggatgtatgg ctatttctgg a 41
<210> 23
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 23
gtccgtacca actagttcgt atccttgcag caacttattc 40
<210> 24
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 24
tgaattcgct ggatcctcgt atccttgcag caacttattc a 41
<210> 25
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 25
gtcgactgga ggatccatgt ggatgtatgg ctatttctgg a 41
<210> 26
<211> 2087
<212> DNA
<213> Oryza sativa
<400> 26
ggaaaggaag aaaaggctaa tatgctcatc ttttttcata gattatactc catatcagta 60
gtatattgtg tcataaagaa taaaagagat atcagactgc cccccctctc tctctcccct 120
ttctcttgca cacaatatca tgatcacact atatttttag tcataggaag agatatttga 180
gaattttgac agcttcctga tcttaggttt ctttattgaa ctgatcttct tttatcagtg 240
ggataaaatg ttgccctata gctatattta caaggacaaa ccaaatgttt tagtatatac 300
cagaatcaaa catgcaacaa ttaattaagt attatagaac taaaaccact ttgttaaaag 360
caaggtctaa attatctgga gaaagtaaga agcaacatgt gatatattat aatattgtct 420
agtttttgta ctaaggtgtg tgttgcaatt gatgcaagtg gggtgtagca taatccatac 480
aagtaagatg ccaagaatgg ggaggagaga ctgtgattat ggcaggaaca tgctcttaat 540
cagtatacag aagtactact actaactact tgcaattact ccaatctctc tcttttctca 600
ttaactgcaa tgcataatcc gtactatccc gtgcaagtaa ctcaaaactt aaggcctcgg 660
ttagggctac taaatgaact atctgcaaat cccgttgttc tcccgttgac aatcatatac 720
ttagcatatt actcattgct tgtttgttag cttatcaagc acatcaaaaa aataaaattt 780
ttaaacttag ttttaagtta tcttgaatca tcgtttattg tcaatattat ctttttgaac 840
cgtcaataaa aaatataaaa aattatctat gaactttcct ttttctgctt cattcttttt 900
tatggcttat cagccatagt tcaaacgatc caccgtagct caaatatcct actactaatt 960
atttttcagc taaaaaagtt agcttccatt ttccaacctt acaatcaagc taacacagtc 1020
actgtcatat aaatagtata ctcaccctaa tcaagctaaa tcttttattt tcctaatgac 1080
tgaactccga aataatatta aattagaaat ctaatgatct agaagatgaa aaccacctct 1140
tttctaatca agctctcttt tgtaaccacc caccaccaca gccatcaaca ccaccaacag 1200
tccaacactg cccaattgcc catgctccag acttctttct actcctacat tccacatatc 1260
tccatggaca gtaactcctc ccaagctacc acttcaaccc taatcccctc tctctctctt 1320
ccgcagaggt agagtgagag agatggtcag atagctagat tgatatccct ctctctctct 1380
cacacacatc tctttttgca agatctcttc ttgttcatca tcttcttctt tttttctccc 1440
ccttttgctt caccaatcca tcttttgtca cgagatgtgg ccgagctgaa gctagtagta 1500
gtggagcagc gaaagcaagt acgccaagaa aaaaaaaagg aagaagaaag aagaaagaaa 1560
gaaagaaaaa aacgccagct tgagggcaga gggcaaaagc ggcgacgagg agcagtggcc 1620
aaagctcaga ttcttcccgt gggctatttt taccacccgc atcccctctc tttgagcccc 1680
ttggccgatt cattcaccga cgcaaagatc caacccctct tcaggtgtcg gcagatgccg 1740
cctttgtgag gtttccagtg gggggatttc tcgtcgtttc ttgcgtgcgg ttgcgttctt 1800
gatccagtga gcgcacggat atatccgccc tggtttagta gagagagaga gagagagaga 1860
gagagagaga gagagaggtt cttgattgag ttccaagtgt tggattgggt tcttggagct 1920
gttggattgg gtttttttgg gagagagatg ggggtttgga ggtgtgtggg ttgaccgaat 1980
tggatcaaga ttattgcggg aggggggggg gggggttgca atggcggatt tggggctgtg 2040
gaagcaaggg tggaggtggg tggtgtccca gaagcacatc ctgacat 2087
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 27
tttgacagct tcctgatctt 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 28
caagtaagat gccaagaatg 20
<210> 29
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 29
ttgtcaacgg gagaacaac 19
<210> 30
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 30
taggatattt gagctacgg 19
<210> 31
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 31
tagaaagaag tctggagca 19
<210> 32
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 32
aacgccagct tgagggcag 19
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 33
ttctcgtcgt ttcttgcgtg 20
<210> 34
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 34
gtgtgtgggt tgaccgaat 19
<210> 35
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 35
ggcattgtca acgggagaac aac 23
<210> 36
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 36
aaacgttgtt ctcccgttga caa 23
<210> 37
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 37
ggcaaacgcc agcttgaggg cag 23
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 38
aaacctgccc tcaagctggc gtt 23
<210> 39
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 39
gccgtttgac agcttcctga tctt 24
<210> 40
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 40
aaacaagatc aggaagctgt caaa 24
<210> 41
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 41
gccgcaagta agatgccaag aatg 24
<210> 42
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 42
aaaccattct tggcatctta ctt 23
<210> 43
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 43
gttgtaggat atttgagcta cgg 23
<210> 44
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 44
aaacccgtag ctcaaatatc cta 23
<210> 45
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 45
gttgtagaaa gaagtctgga gca 23
<210> 46
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 46
aaactgctcc agacttcttt cta 23
<210> 47
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 47
tcagttctcg tcgtttcttg cgtg 24
<210> 48
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 48
aaaccacgca agaaacgacg agaa 24
<210> 49
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 49
tcaggtgtgt gggttgaccg aat 23
<210> 50
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 50
aaacattcgg tcaacccaca cac 23
<210> 51
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 51
ggaaaggaag aaaaggctaa tatgctcatc 30
<210> 52
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 52
atgtcaggat gtgcttctgg gacac 25
<210> 53
<211> 1344
<212> DNA
<213> Oryza sativa
<400> 53
ggaaaggaag aaaaggctaa tatgctcatc ttttttcata gattatactc catatcagta 60
gtatattgtg tcataaagaa taaaagagat atcagactgc cccccctctc tctctcccct 120
ttctcttgca cacaatatca tgatcacact atatttttag tcataggaag agatatttga 180
gaattttgac agcttcctga tggtttcttt attgaactga tcttctttta tcagtgggat 240
aaaatgttgc cctatagcta tatttacaag gacaaaccaa atgttttagt atataccaga 300
atcaaacatg caacaattaa ttaagtatta tagaactaaa accactttgt taaaagcaag 360
gtctaaatta tctggagaaa gtaagaagca acatgtgata tattataata ttgtctagtt 420
tttgtactaa ggtgtgtgtt gcaattgatg caagtggggt gtagcataat ccatacaagt 480
aagatgccaa gatccagact tctttctact cctacattcc acatatctcc atggacagta 540
actcctccca agctaccact tcaaccctaa tcccctctct ctctcttccg cagaggtaga 600
gtgagagaga tggtcagata gctagattga tatccctctc tctctctcac acacatctct 660
ttttgcaaga tctcttcttg ttcatcatct tcttcttttt ttctccccct tttgcttcac 720
caatccatct tttgtcacga gatgtggccg agctgaagct agtagtagtg gagcagcgaa 780
agcaagtacg ccaagaaaaa aaaaggaaga agaaagaaga aagaaagaaa gaaaaaaacg 840
ccagcttgag ggacagaggg caaaagcggc gacgaggagc agtggccaaa gctcagatcc 900
ttcccgtggg ctatttttac cacccgcatc ccctctcttt gagccccttg gccgattcat 960
tcaccgacgc aaagatccaa cccctcttca ggtgtcggca gatgccgcct ttgtgaggtt 1020
tccagtgggg ggatttctcg tcgtttcttg cgtgcggttg cgttcttgat ccagtgagcg 1080
cacggatata tccgccctgg tttagtagag agagagagag agagagagag agagaggttc 1140
ttgattgagt tccaagtgtt ggattgggtt cttggagctg ttggattggg tttttttggg 1200
agagagatgg gggtttggag gtgtgtgggt tgaccaattg gatcaagatt attgcgggag 1260
ggggggggga ggttgcaatg gcggatttgg ggctgtggaa gcaagggtgg aggtgggtgg 1320
tgtcccagaa gcacatcctg acat 1344
<210> 54
<211> 981
<212> DNA
<213> Oryza sativa
<400> 54
ggaaaggaag aaaaggctaa tatgctcatc ttttttcata gattatactc catatcagta 60
gtatattgtg tcataaagaa taaaagagat atcagactgc cccccctctc tctctcccct 120
ttctcttgca cacaatatca tgatcacact atatttttag tcataggaag agatatttga 180
gaattttgac agcttcctga tattgtgtga tcatgatatc aggtttcttt attgaactga 240
tcttctttta tcagtgggat aaaatgttgc cctatagcta tatttacaag gacaaaccaa 300
atgttttagt atataccaga atcaaacatg caacaattaa ttaagtatta tagaactaaa 360
accactttgt taaaagcaag gtctaaatta tctggagaaa gtaagaagca acatgtgata 420
tattataata ttgtctagtt tttgtactaa ggtgtgtgtt gcaattgatg caagtggggt 480
gtagcataat ccatacaagt aagatgccaa gaccctcaag ctggcgtttt tttctttctt 540
tctttcttct ttcttcttcc tttttttttc ttggcgtact tgctttcgct gctccactac 600
tactagcttc agctcggcca catctcgtga caaaagatgg attggtgaag caaaaggggg 660
agaaaaaaag aagaagatga tgaacaagaa gagatcttgc aaaaagagat gtgtgtgaga 720
gagagagagg gatatcaatc tagctatctg accatctctc tcactctacc tctgcggaag 780
agagagagag gggattaggg ttgaagtggt agcttgggag gagttactgt ccatggagat 840
atgtggaatg taggagtaga aagaagtctg gagaattgga tcaagattat tgcgggaggg 900
gggggggggt tgcaatggcg gatttggggc tgtggaagca agggtggagg tgggtggtgt 960
cccagaagca catcctgaca t 981
<210> 55
<211> 528
<212> PRT
<213> Brachypodium_distachyon
<400> 55
Met Asp Ile Met Thr Trp Ala His Met Ala Ala Gly Cys Gly Arg Glu
1 5 10 15
Arg Val Ala Ser Leu Val Asp Arg His Trp Pro Ala Val Ser Arg Ala
20 25 30
Cys Val Cys Ser Ser Cys Phe Val Leu Ala Ala Leu Arg Gln Trp Gln
35 40 45
Gly Cys Thr Ala Arg Gly Phe Leu Gly Leu Ala Ser Leu Gly Pro Ala
50 55 60
Ala Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Met Thr Ser Pro
65 70 75 80
Val Cys Ala Leu Tyr Ala Leu Leu Ile Leu Gly Ala Thr Gly Ala Val
85 90 95
Ile His Tyr Met Gly Tyr Thr Pro Gly Leu Leu Ile Val Gly Leu Phe
100 105 110
Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly Met
115 120 125
Leu Leu Val Ala Gly Gly Ser Met Cys Ser Leu Lys His Ala Arg Phe
130 135 140
Val Ile Pro Val Leu Ala Val Tyr Ala Val Tyr Cys Val Ala Val Arg
145 150 155 160
Val Gly Trp Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu Thr
165 170 175
Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Gly Ser Thr Glu
180 185 190
Glu Met Glu Phe Glu Glu Met Lys Asp Pro His Pro Gly Met Asp Glu
195 200 205
Phe Tyr Pro Ser Tyr Glu Tyr Pro Pro Ala Pro Asp Ser Glu Pro Glu
210 215 220
Thr Val Ser Ser Ala Lys Pro Phe Cys Ala Ser Pro Thr Gln Asp Val
225 230 235 240
Leu His Val Gln Lys Glu Ala Ser Pro Ser Lys Ile Val Lys Ser Asp
245 250 255
Ser Thr Ala Leu Asp Glu Met Lys Arg Ile Met Asp Gly Ser Thr Tyr
260 265 270
Tyr Glu Ile Phe Gly Ile Pro Arg Asn Arg Ser Ala Asp Leu Lys Ile
275 280 285
Leu Lys Gly Glu Tyr Arg Arg Met Ala Met Leu Val His Pro Asp Lys
290 295 300
Asn Met Gly Asn Ser Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln Ser
305 310 315 320
Ala Tyr Glu Val Leu Ser Asp Leu Thr Lys Lys Asn Ser Tyr Asp Glu
325 330 335
Gln Leu Arg Lys Glu Glu Ser Arg Gln Met Thr Gln Arg Ser Arg Val
340 345 350
Val Ser Gln Gln Ser Gly Val Glu Phe Leu Ser Glu Glu Ser Arg Arg
355 360 365
Ile Gln Cys Thr Lys Cys Gly Asn Phe His Leu Trp Ile Cys Thr Lys
370 375 380
Arg Ser Lys Ala Lys Ala Arg Trp Cys Gln Asp Cys Ser Gln His His
385 390 395 400
Val Ala Lys Asp Gly Asp Gly Trp Val Glu Asn Gly Tyr Ser Thr Ser
405 410 415
Leu Lys Ile Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys Ile
420 425 430
Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys Pro
435 440 445
Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Ala Asp Arg
450 455 460
Met Pro Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asp Ala
465 470 475 480
Glu Met Ile Pro Glu Asp Glu Phe Asp Leu Trp Leu Gln Gln Ala Leu
485 490 495
Ala Thr Gly Val Phe Ser Asp Ser Pro Lys Arg Arg Lys Ser Trp Ser
500 505 510
Pro Phe Lys Leu Thr Gln Lys Gly Val Arg Ser Trp Arg Arg Ser Ser
515 520 525
<210> 56
<211> 545
<212> PRT
<213> Hordeum_vulgare
<400> 56
Met Ala Gly Leu Gly Leu Trp Asn Gln Gly Trp Thr Trp Val Leu Ser
1 5 10 15
Gln Lys His Val Val Ala Trp Ala His Ala Ala Ala Gly Cys Gly Arg
20 25 30
Asp Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Ala Thr Ser Ser Arg Leu Val Leu Glu Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Thr Ala Arg Gly Leu Leu Ala Leu Ala Ser Leu Gly Pro
65 70 75 80
Ala Ala Val Phe Val Ile Leu Trp Ser Cys Phe Val Cys Met Thr Ser
85 90 95
Ser Ala Cys Ala Leu Tyr Ala Leu Leu Ala Leu Gly Ala Val Gly Ala
100 105 110
Val Ile His Tyr Met Gly Tyr Thr Pro Gly Leu Leu Ile Val Gly Leu
115 120 125
Phe Gly Ile Met Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Val Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Phe Val Ile Pro Val Leu Ala Met Tyr Ala Val Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Trp Leu Gly Val Phe Phe Met Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Arg Pro Phe Glu Glu Met Lys Asp Ser Asp Pro Ala Thr Asp
210 215 220
Ala Phe Phe Arg Gly Cys Glu Tyr Pro Pro Ala Pro Glu Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Phe Cys Ala Ala Pro Thr Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Pro Ser Pro Thr Lys Ile Val Lys Ser
260 265 270
Asn Ser Thr Ser Leu Asp Glu Met Lys Arg Ile Met Asp Gly Ser Thr
275 280 285
Tyr Tyr Glu Val Leu Gly Ile Pro Arg Ser Lys Ser Ile Asn Gln Ile
290 295 300
Glu Leu Lys Lys Glu Tyr Arg Lys Leu Ala Val Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Phe Glu Val Leu Ser Asp Leu Thr Lys Lys Asn Gly Tyr Asp
340 345 350
Glu Gln Leu Arg Lys Glu Glu Ser Arg Gln Met Thr Gln Arg Ser Arg
355 360 365
Val Val Ser Gln Pro Ser Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Leu Trp Ile Cys Thr
385 390 395 400
Lys Arg Ser Lys Ala Lys Ala Arg Trp Cys Gln Glu Cys Ser Gln Tyr
405 410 415
His Val Ala Lys Asp Gly Asp Gly Trp Val Glu Asn Arg Tyr Ser Thr
420 425 430
Ser Leu Lys Ile Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys
435 440 445
Ile Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys
450 455 460
Pro Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Ala Asp
465 470 475 480
Arg Met Pro Gln Arg Ser His Ser Ser Arg Tyr Pro Phe Ser Leu Asp
485 490 495
Ala Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala
500 505 510
Leu Ala Thr Gly Val Phe Ser Asp Ser Pro Lys Arg Arg Lys Ser Trp
515 520 525
Ser Pro Phe Lys Leu Pro Gln Lys Gly Ile Arg Ser Trp Arg Arg Ser
530 535 540
Ser
545
<210> 57
<211> 545
<212> PRT
<213> Oryza_brachyantha
<400> 57
Met Ala Asp Leu Gly Leu Trp Lys Gln Gly Trp Arg Trp Val Leu Ser
1 5 10 15
Gln Lys His Ile Leu Thr Trp Ala His Met Ala Ala Ser Gly Gly Thr
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Thr Cys Val Ser Ser Gly Arg Leu Ala Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Ala Ala Arg Gly Ile Leu Glu Met Ala Ser Leu Gly Pro
65 70 75 80
Ala Ser Val Phe Val Ile Leu Trp Ser Cys Phe Val Cys Met Thr Ser
85 90 95
Pro Ala Cys Ala Leu Tyr Ala Leu Leu Ser Leu Gly Ala Ala Gly Ala
100 105 110
Val Ile His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Ile Ser Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Phe Val Ile Pro Val Leu Ala Met Tyr Ala Val Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Leu Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Met Asn Lys Leu Leu Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Arg Gln Phe Glu Glu Thr Lys His Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Tyr Pro Thr Ala Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Phe Cys Ser Thr Pro Val Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Ala Ser Pro Ser Lys Val Val Lys Ser
260 265 270
Asp Ser Val Ser Leu Asp Glu Met Lys Arg Ile Met Asp Gly Leu Thr
275 280 285
His Tyr Glu Val Leu Gly Ile Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Thr Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Thr Tyr Asp
340 345 350
Asp Gln Leu Arg Lys Glu Glu Ser Arg Lys Met Thr Gln Arg Ser Arg
355 360 365
Val Val Ser Gln Gln Thr Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Leu Trp Ile Cys Thr
385 390 395 400
Lys Lys Ser Lys Ala Lys Ala Arg Trp Cys Gln Asp Cys Ser Asp Phe
405 410 415
His Pro Ala Lys Asp Gly Asp Gly Trp Val Glu Asn Lys Phe Ser Ala
420 425 430
Ser Phe Lys Met Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys
435 440 445
Ile Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys
450 455 460
Pro Asn Thr His Gly Pro Ser Phe Met Val Asn Met Val Gly Ala Asp
465 470 475 480
Arg Met Ser Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asn
485 490 495
Ala Glu Met Val Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala
500 505 510
Leu Ala Ser Gly Val Phe Ala Asp Ser Pro Lys Arg Arg Lys Ser Trp
515 520 525
Ser Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser
530 535 540
Ser
545
<210> 58
<211> 545
<212> PRT
<213> Panicum_hallii
<400> 58
Met Ala Asp Leu Gly Leu Trp Lys Gln Ala Trp Arg Trp Val Leu Ser
1 5 10 15
Gln Lys His Ile Leu Ala Trp Ala His Thr Ala Ala Cys Gly Ser Arg
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Ala Thr Ser Ser Arg Leu Ala Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Met Ala Arg Gly Val Leu Ala Val Ala Ser Leu Gly Pro
65 70 75 80
Ala Ala Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Met Thr Ser
85 90 95
Pro Ala Trp Ala Leu Phe Ala Leu Leu Ser Leu Gly Ala Ala Gly Ala
100 105 110
Val Val His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Val Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Tyr Val Ile Pro Ile Leu Thr Thr Tyr Ala Ile Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Trp Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Gly Cys Thr
195 200 205
Glu Glu Glu Gln Phe Glu Asp Met Lys Asp Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Phe Pro Pro Thr Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Tyr Cys Ser Ala Pro Thr Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Glu Pro Pro Ser Lys Val Val Lys Ser
260 265 270
Asp Ser Ser Ser Leu Asp Glu Ile Lys Arg Ile Met Asp Gly Ser Asn
275 280 285
Tyr Tyr Glu Val Leu Gly Ile Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Ser Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Ser Tyr Asp
340 345 350
Glu Gln Leu Arg Lys Glu Glu Ser Gln Asn Met Thr Pro Arg Ser Arg
355 360 365
Val Val Ser Gln Gln Ser Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Ile Trp Ile Cys Thr
385 390 395 400
Lys Arg Ser Lys Thr Lys Ala Arg Phe Cys Gln Gly Cys Asp Gln Phe
405 410 415
His Gln Ala Lys Asp Gly Asp Gly Trp Val Glu Thr Arg Phe Ser Thr
420 425 430
Ser Val Lys Met Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys
435 440 445
Ile Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys
450 455 460
Pro Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Ala Asp
465 470 475 480
Arg Met Pro Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asp
485 490 495
Ala Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala
500 505 510
Leu Ala Ser Gly Val Phe Ala Asp Ser Pro Lys Arg Arg Lys Ser Trp
515 520 525
Ser Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser
530 535 540
Ser
545
<210> 59
<211> 544
<212> PRT
<213> Setaria_italica
<400> 59
Met Ala Asp Leu Gly Leu Trp Lys Gln Ala Trp Arg Trp Val Leu Ser
1 5 10 15
Gln Lys His Ile Leu Ala Trp Ala His Thr Ala Ala Cys Gly Ser Arg
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Ala Thr Ser Ser Arg Leu Ala Leu Ala Ala Leu Leu Gln Trp
50 55 60
Arg Gly Cys Met Ala Arg Gly Val Leu Ala Val Ala Ser Leu Gly Pro
65 70 75 80
Ala Ala Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Met Thr Ser
85 90 95
Pro Ala Trp Ala Leu Phe Ala Leu Leu Leu Leu Gly Ala Ala Gly Ala
100 105 110
Val Val His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Val Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Tyr Val Ile Pro Ile Leu Thr Thr Tyr Ala Ile Tyr Cys Val Ala Ile
165 170 175
Arg Val Gly Trp Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Ala Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Glu Ser Thr
195 200 205
Glu Glu Lys Phe Glu Asp Met Lys Asp Ser Asp Pro Val Met Asp Glu
210 215 220
Phe Tyr Arg Ser Cys Glu Phe Pro Pro Ala Pro Asp Ser Glu Pro Glu
225 230 235 240
Thr Val Ser Ser Ala Lys Pro Tyr Cys Ser Ser Pro Thr Gln Asp Val
245 250 255
Leu His Val Gln Lys Glu Glu Pro Pro Ser Lys Val Val Lys Ser Asp
260 265 270
Ser Ser Ser Leu Asp Glu Ile Lys Arg Ile Met Asp Gly Ser Asn His
275 280 285
Tyr Glu Val Leu Gly Ile Pro Arg Asn Arg Ser Ile Asp Gln Lys Ser
290 295 300
Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp Lys
305 310 315 320
Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln Ser
325 330 335
Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Arg Asn Ser Tyr Asp Glu
340 345 350
Gln Leu Arg Lys Glu Glu Ser Gln Lys Met Thr Pro Arg Ser Arg Val
355 360 365
Val Ser Gln Gln Gly Gly Val Glu Phe Leu Ser Glu Glu Ser Arg Arg
370 375 380
Ile Gln Cys Thr Lys Cys Gly Asn Phe His Ile Trp Ile Cys Thr Lys
385 390 395 400
Arg Ser Lys Thr Lys Ala Arg Phe Cys Gln Gly Cys Asp Gln Tyr His
405 410 415
Gln Ala Lys Asp Gly Asp Gly Trp Val Glu Thr Arg Phe Ser Thr Ser
420 425 430
Tyr Lys Met Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys Ile
435 440 445
Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys Pro
450 455 460
Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Ala Asp Arg
465 470 475 480
Met Pro Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asp Ala
485 490 495
Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala Leu
500 505 510
Ala Ser Gly Val Phe Ala Asp Ser Pro Lys Arg Arg Lys Ser Trp Ser
515 520 525
Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser Ser
530 535 540
<210> 60
<211> 545
<212> PRT
<213> Zea_mays
<400> 60
Met Glu Asp Leu Gly Leu Trp Asn Gln Ala Trp Met Trp Val Leu Ser
1 5 10 15
Gln Lys His Ile Leu Ala Trp Ala His Thr Ala Ala Cys Gly Ser Arg
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Gly Cys Ala Thr Ser Ser Arg Leu Thr Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Met Ala Arg Gly Val Leu Ala Val Ala Ser Leu Gly Pro
65 70 75 80
Ala Ala Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Met Thr Ser
85 90 95
Pro Ala Cys Ala Leu Tyr Ala Leu Leu Ser Leu Gly Ala Ala Ala Ala
100 105 110
Val Val His Tyr Met Gly Tyr Thr Pro Gly Leu Leu Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Val Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Tyr Val Thr Pro Val Leu Thr Ser Tyr Ala Ile Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Trp Leu Gly Val Phe Leu Thr Phe Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Ala Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Ser Gln Phe Glu Asp Met Lys Asp Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Phe Pro Ser Val Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Tyr Cys Ser Ala Pro Ile Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Glu Pro Pro Ser Lys Ile Val Lys Ser
260 265 270
Asp Ser Ser Ser Ser Asp Glu Ile Lys Arg Ile Met Asp Gly Ser Asn
275 280 285
His Tyr Glu Val Leu Gly Val Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Ala Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Ser Tyr Asp
340 345 350
Gln Gln Leu Arg Lys Glu Glu Ser Gln Lys Met Thr Pro Arg Ser Arg
355 360 365
Ala Val Ser Gln Gln Ser Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Ile Trp Ile Cys Thr
385 390 395 400
Lys Arg Ser Lys Thr Lys Ala Arg Phe Cys Gln Gly Cys Asp Gln Phe
405 410 415
His Gln Ala Lys Asp Gly Asp Gly Trp Val Glu Thr Arg Phe Ser Ser
420 425 430
Ser Ile Lys Met Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys
435 440 445
Ile Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys
450 455 460
Pro Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Ala Asp
465 470 475 480
Arg Met Pro Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asp
485 490 495
Ala Glu Met Ile Pro Asp Asp Glu Phe Glu Met Trp Leu Gln Gln Ala
500 505 510
Leu Ala Ser Gly Val Phe Ala Asp Ser Pro Lys Arg Arg Lys Ser Trp
515 520 525
Ser Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser
530 535 540
Ser
545
<210> 61
<211> 545
<212> PRT
<213> Sorghum_bicolor
<400> 61
Met Ala Asp Leu Gly Leu Trp Lys Gln Ala Trp Met Trp Val Leu Ser
1 5 10 15
Gln Lys His Ile Leu Ala Trp Ala His Thr Ala Ala Cys Gly Ser Arg
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Ala Thr Ser Ser Arg Leu Ala Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Thr Ala Arg Gly Val Leu Ala Val Ala Ser Leu Gly Pro
65 70 75 80
Ala Ala Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Met Thr Ser
85 90 95
Pro Ala Cys Ala Leu Tyr Ala Leu Leu Ser Leu Gly Ala Ala Ala Ala
100 105 110
Val Val His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Val Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Tyr Val Ile Pro Val Leu Thr Ser Tyr Ala Ile Tyr Ser Val Ala Val
165 170 175
Arg Val Gly Trp Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Ala Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Ser Gln Phe Glu Asp Ile Lys Gly Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Phe Pro Pro Val Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Tyr Cys Thr Ala Pro Val Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Glu Pro Pro Ser Lys Val Val Lys Ser
260 265 270
Asp Ser Ser Ser Leu Asp Glu Ile Lys Arg Ile Met Asp Gly Ser Asn
275 280 285
His Tyr Glu Val Leu Gly Val Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Thr Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Ser Tyr Asp
340 345 350
Glu Gln Leu Arg Lys Glu Glu Ser Leu Lys Met Thr Pro Arg Ser Arg
355 360 365
Val Val Ser Gln Gln Ser Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Ile Trp Ile Cys Thr
385 390 395 400
Lys Arg Ser Lys Thr Arg Ala Arg Phe Cys Gln Gly Cys Asp Gln Phe
405 410 415
His Gln Ala Lys Asp Gly Asp Gly Trp Val Glu Thr Arg Phe Ser Ser
420 425 430
Ser Ile Lys Met Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys
435 440 445
Ile Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys
450 455 460
Pro Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Thr Asp
465 470 475 480
Arg Met Pro Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asp
485 490 495
Ala Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala
500 505 510
Leu Ala Ser Gly Val Phe Ala Asp Ser Pro Lys Arg Arg Lys Ser Trp
515 520 525
Ser Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser
530 535 540
Ser
545

Claims (6)

1. A method for controlling rice seed width by knocking out or inhibiting expression of a gene controlling rice seed width, characterized in that a nucleotide sequence of the gene controlling rice seed width is selected from one of the sequences of the following group:
(a) As set forth in SEQ ID NO: 1. 2, 20 or 21;
(b) Encoding the sequence as shown in SEQ ID NO:3, and a nucleotide sequence of the amino acid sequence shown in 3.
2. The method according to claim 1, wherein the rice material having a large grain phenotype is obtained by mutating a nucleotide sequence of a gene regulating rice seed width selected from one of the following sequences:
(a) As set forth in SEQ ID NO: 1. 2, 20 or 21;
(b) Encoding the sequence as shown in SEQ ID NO:3, and a nucleotide sequence of the amino acid sequence shown in 3.
3. Use of the method according to any one of claims 1-2 for regulating grain width of rice seeds.
4. The application of the expression cassette, the expression vector and the engineering bacteria in regulating the grain width of rice seeds is characterized in that the expression cassette, the expression vector and the engineering bacteria comprise genes for regulating the grain width of rice seeds, and the nucleotide sequence of the genes for regulating the grain width of rice seeds is selected from one of the following sequences:
(a) As set forth in SEQ ID NO: 1. 2, 20 or 21;
(b) Encoding the sequence as shown in SEQ ID NO:3, and a nucleotide sequence of the amino acid sequence shown in 3.
5. A method for obtaining a mutant material, wherein the mutant material is caused by a mutation of a gene regulating rice seed width, a plant containing the mutated nucleotide sequence has a phenotype of rice seed width, and the nucleotide sequence of the gene regulating rice seed width is selected from one of the following sequences:
(a) As set forth in SEQ ID NO: 1. 2, 20 or 21;
(b) Encoding the sequence as shown in SEQ ID NO:3, a nucleotide sequence of the amino acid sequence shown in 3;
wherein the mutation is site-directed mutagenesis, and the nucleotide sequence after the mutation is shown in SEQ ID NO:4, the coded amino acid sequence is shown as SEQ ID NO: shown at 5.
6. The use of the mutant material obtained by the method of claim 5 for controlling rice grain width by controlling rice grain width gene or as a molecular marker for identifying large and small varieties of crops.
CN201910752694.7A 2018-01-24 2019-08-13 Gene for regulating and controlling plant seed size and application thereof Active CN110373418B (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CN201810067473 2018-01-24
CN2019100515250 2019-01-21
CN201910051525.0A CN110079532A (en) 2018-01-24 2019-01-21 Regulate and control gene and its application of size of plant seed

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CN110373418A CN110373418A (en) 2019-10-25
CN110373418B true CN110373418B (en) 2024-05-10

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CN109355291B (en) * 2018-11-22 2022-01-18 深圳市作物分子设计育种研究院 Identification and application of plant endosperm specific expression promoter pOsEnS93
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010236A1 (en) * 1991-11-15 1993-05-27 The University Of Melbourne Protein allergens of the species cynodon dactylon
CN101161675A (en) * 2006-10-13 2008-04-16 中国科学院上海生命科学研究院 Rice big grain gene and uses thereof
CN107630031A (en) * 2012-11-09 2018-01-26 深圳市作物分子设计育种研究院 A kind of method and system of regulation and control plant fertility
CN108441499A (en) * 2017-02-16 2018-08-24 深圳兴旺生物种业有限公司 Male fertile related gene HT2925 and its application
CN108823207A (en) * 2018-06-25 2018-11-16 中国农业科学院麻类研究所 A kind of Bn-miR43 of ramie and its application

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013188578A1 (en) * 2012-06-12 2013-12-19 The Johns Hopkins University Efficient, expansive, user-defined dna mutagenesis

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010236A1 (en) * 1991-11-15 1993-05-27 The University Of Melbourne Protein allergens of the species cynodon dactylon
CN101161675A (en) * 2006-10-13 2008-04-16 中国科学院上海生命科学研究院 Rice big grain gene and uses thereof
CN107630031A (en) * 2012-11-09 2018-01-26 深圳市作物分子设计育种研究院 A kind of method and system of regulation and control plant fertility
CN108441499A (en) * 2017-02-16 2018-08-24 深圳兴旺生物种业有限公司 Male fertile related gene HT2925 and its application
CN108823207A (en) * 2018-06-25 2018-11-16 中国农业科学院麻类研究所 A kind of Bn-miR43 of ramie and its application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DNAJ heat shock N-terminal domain-containing protein, putative, expressed [Oryza sativa Japonica Group];Buell C.R. et al;Genbank;20110505;第1-2页 *
水稻粒宽基因GS5的调控与分子机理研究;许纯钰;《中国博士学位论文全文数据库 基础科学辑》;20160115(第1期);第A006-68页 *

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