WO2014131348A1 - Promoter having high-efficiency initiating activity - Google Patents

Promoter having high-efficiency initiating activity Download PDF

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WO2014131348A1
WO2014131348A1 PCT/CN2014/072540 CN2014072540W WO2014131348A1 WO 2014131348 A1 WO2014131348 A1 WO 2014131348A1 CN 2014072540 W CN2014072540 W CN 2014072540W WO 2014131348 A1 WO2014131348 A1 WO 2014131348A1
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polynucleotide
seq
gene
promoter
nucleotide sequence
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PCT/CN2014/072540
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French (fr)
Chinese (zh)
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方玉达
夏溪
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中国科学院上海生命科学研究院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells

Definitions

  • the present invention is in the field of biotechnology and botany; more specifically, the present invention relates to a promoter having efficient driving activity. Background technique
  • Promoters commonly used in plant genetic engineering are classified into three types according to their functions and functions: constitutive promoters, inducible promoters, and tissue-specific promoters. Among them, constitutive promoters are most commonly used.
  • the constitutive promoter is divided into two major categories: endogenous constitutive and heterologous constitutive promoter. Endogenous promoters commonly used in plant genetic engineering mainly include the promoters of the rice actin gene and the ubiquitin gene.
  • Heterologous constitutive promoters include the Nos and Ocs promoters from Agrobacterium and the CaMV 35S promoter derived from the Tobacco Mosaic Virus gene. The constitutive promoter is expressed in all tissues and is persistent but not spatiotemporal specific.
  • Histone Proteins that bind to DNA in the nucleus of eukaryotes include histones and non-histones.
  • Histone refers to a general term for basic proteins that bind to DNA in the nucleus. Histones are rich in basic amino acids such as arginine and lysine, which add up to about 1/4 of all amino acid residues. They have a small molecular weight of about 10 000 to 20 000 Daltons. There are five major histones - Hl, H2A, H2B, H3, H4, which have different molecular weights and amino acid compositions.
  • the four histones (H2A, H2B, H3, H4) have a small molecular weight (102 to 135 amino acid residues), and the molecular weight of HI is large (about 220 amino acid residues), and the sequence is conservative.
  • the degree is relatively low, but the structure is similar.
  • the spherical central domain of HI histone is evolutionarily conserved, while the amino acids of the two "arms" at the N-terminus and C-terminus are larger, so HI is not as evolutionary as nucleosomal histone. So conservative, HI acts as a linker when it forms a nucleosome, which imparts polarity to chromatin.
  • histones In addition to conventional histones, histones also have a variety of variants, particularly in the core histones H2A, H3 and linked histone HI.
  • the core histones H2B and H4 vary relatively little.
  • Histone variants can be divided into two categories depending on the amino acid sequence of the corresponding conventional histones: one is a variant produced by amino acid substitution, and the total number of amino acids is unchanged, called Homomorphous variants, such as plants.
  • Conventional histone H3 ie H3.1
  • H3.3 There are only four amino acids in between, which are in the 31st position (glycine in H3.1, threonine in H3.3) and 41 (phenylalanine in H3.1, H3.
  • H3.1 is tyrosine
  • 87 cyste in H3.1, histidine in H3.3
  • 90 glycine in H3.1, leucine in H3.3
  • H3.1 is mainly loaded into chromatin during DNA replication
  • H3.3 can be loaded into interphase staining independent of DNA replication.
  • Quality. H3.1 is primarily associated with silent chromatin, whereas H3.3 is primarily involved in transcriptionally active chromatin.
  • the histone H3.3 of the plants was found to be very conserved in the results of amino acid sequence analysis of plants including Arabidopsis thaliana, rice and maize histone H3.3.
  • an isolated polynucleotide is provided, said polynucleotide being:
  • the nucleotide sequence is capable of hybridizing under stringent conditions to any of the polynucleotide sequences defined in (1) to (10) and has
  • the nucleotide sequence is 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more; more preferably) of the polynucleotide sequence defined by any one of (1) to (10) Preferably, 99% or more of the polynucleotides having the same identity and having the polynucleotide function defined by any of (1) to (10).
  • (4) comprising: a polynucleotide of the nucleotide sequence shown in position 464-1405 (pHTR4-5 (942 bp)) of SEQ ID NO: 34; SEQ ID NO: 34 A polynucleotide of a nucleotide sequence shown in position 276-1405 (pHTR4-6 (1130 bp)); or a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 34.
  • (5) comprising: a polynucleotide of the nucleotide sequence shown at positions 201-521 (pHTR711-2 (521 bp)) in SEQ ID NO: 3; SEQ ID NO: 35 a polynucleotide of a nucleotide sequence shown in position 624-1369 (pHTR711-4 (746 bp)); a nucleoside represented by position 416-1369 (pHTR711-5 (954 bp)) in SEQ ID NO: 35 a polynucleotide of an acid sequence; a polynucleotide of a nucleotide sequence shown at position 200-1369 (pHTR711-6 (1170 bp)) in SEQ ID NO: 35; or a nucleotide represented by SEQ ID NO: Sequence polynucleotide.
  • the use of the polynucleotide is provided for use as a promoter element.
  • the promoter element is used to direct strong expression of the gene of interest.
  • the gene of interest is a structural gene. In another preferred embodiment, the gene of interest encodes a protein having a specific function.
  • the gene of interest is a foreign gene.
  • a vector comprising the polynucleotide as a promoter element.
  • the vector further comprises a gene of interest operably linked to the polynucleotide.
  • the gene of interest is located downstream of the polynucleotide.
  • the gene of interest is located downstream of the polynucleotide and is less than 1000 bp from the polynucleotide; preferably, less than 500 bp; more preferably, less than 200 bp.
  • the gene of interest is located downstream of the polynucleotide and is less than 100 bp, more preferably less than 50 bp from the polynucleotide.
  • the vector is a eukaryotic expression vector.
  • a multiple cloning site or at least one cleavage site is included downstream of the polynucleotide.
  • a genetically engineered host cell is provided (preferably, said host cell is not an animal or plant propagation material), said cell:
  • A2 pCambial300-pHTRl-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in Arabidopsis rosette leaves, stem leaves and inflorescences;
  • B l, B2 pCambial300-pHTR4-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in Arabidopsis rosette leaves, stem leaves and inflorescences;
  • A pCambial300-CaMV35S-GUS transformed flower callus, GUS histochemical staining results
  • B pCambial300-pHTR711-GUS transformed flower 11 callus, GUS histochemical staining results
  • C pCambial300-pHTR712-GUS transformation Zhonghua 11 callus, GUS histochemical staining results.
  • Figure 6. Mapping of rice gene HTR711 and HrR7J2 promoters driving GFP gene expression at the subcellular level.
  • Rice gene HTR711 and HTR712 promoter drive the expression of GFP gene in the nucleus of Trifolium repens leaves;
  • FIG. 7 Schematic diagram of seven Arabidopsis thaliana H7 W promoter fragments.
  • pHTR4-l (116 bp) represents position 548-663 in the sequence of SEQ ID NO: 2; pHTR4-2 (263 bp) represents position 401-663 in the sequence of SEQ ID NO: 2; pHTR4-3 (453 bp) represents SEQ ID NO: position 21-263 in the 2 sequence; pHTR4-4 (663 bp) is the sequence of SEQ ID NO: 2; pHTR4-5 (942 bp) sequence is as position 464-1405 in SEQ ID NO: 34; pHTR4-6 ( The 1130 bp) sequence is located at positions 276-1405 of SEQ ID NO: 34; the pHTR4-7 (1405 bp) sequence is SEQ ID NO: 34 sequence.
  • FIG. 8 Schematic diagram of seven rice gene HTR711 promoter fragments.
  • pHTR711-1 (201 bp) represents position 321-521 of the sequence of SEQ ID NO: 3; pHTR711-2 (321 bp) represents position 201-521 of the sequence of SEQ ID NO: 3; pHTR711-3 (521 bp) represents SEQ ID NO: 3 sequence; pHTR711-4 (746 bp) sequence is located at positions 624-1369 of SEQ ID NO: 35; pHTR711-5 (954 bp) sequence is at positions 416-1369 of SEQ ID NO: 35; pHTR711-6 ( The 1170 bp) sequence is in positions 200-1369 of SEQ ID NO: 35; the pHTR711-7 sequence is in SEQ ID NO: 35 sequence.
  • E Fluorescence of the pHTR4-5 promoter
  • F Fluorescence map of the pHTR4-6 promoter
  • G Fluorescence of the pHTR4-7 promoter.
  • Figure 10 Fluorescence of the seven rice gene HrR7J promoter.
  • G Fluorescence of the pHTR711-7 promoter.
  • Figure 11. Box diagram of the fluorescence values of the seven Arabidopsis gene HTR4 and seven rice gene HTR711 promoters.
  • FIG. 1 B: Box diagram of the fluorescence values of seven Arabidopsis gene HrR7 promoters.
  • Arabidopsis thaliana gene leg 1, ⁇ 4 promoter and CaMV35S promoter drive Gt/S gene expression map.
  • A2 pCambial300-pHTR101-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in Arabidopsis stem leaves and rosette leaves;
  • Bl, B2 P Cambial300-pHTR102-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in Arabidopsis stem leaves and rosette leaves;
  • A pCambial300-pHTR706-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in stem leaves;
  • B P Cambial300-pHTR706-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in rosette leaves.
  • the present inventors have revealed, through extensive and intensive research, a nucleic acid which is used as a promoter element to direct high-level expression of a gene of interest.
  • the promoter of the present invention is derived from the promoter region of the Hist one 3.3 gene.
  • the promoter of the present invention has a wide range of activities and is capable of directing high level expression (strong expression) of the gene of interest.
  • the present invention has been completed on this basis.
  • promoter or “promoter region (domain)” refers to a nucleic acid sequence that is normally present upstream (5' end) of the coding sequence of the gene of interest and is capable of directing transcription of the nucleic acid sequence into mRNA. .
  • the promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.
  • the promoter or promoter region includes a variant of a promoter which is obtained by inserting or deleting a regulatory region, performing random or site-directed mutagenesis or the like.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • operably linked refers to a spatial arrangement of the functionality of two or more nucleic acid regions or nucleic acid sequences.
  • the promoter region is placed at a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region such that the promoter region is “operably linked” to the nucleic acid sequence.
  • high level expression refers to: using the promoter of the present invention to direct expression of a gene of interest (such as the GUS gene) and using the promoter of the present invention as compared to the expression of the CaMV35S promoter to direct the gene of interest.
  • the amount of expression obtained is significantly higher. Detection of the amount of protein expression is a technique well known to those skilled in the art.
  • nucleotide sequence shown by (1-743) to 1405 in SEQ ID NO: 34 means that the sequence can start from position 1 in SEQ ID NO: 34 to The base of any of position 743 ends at the 1405th base of SEQ ID NO: 1.
  • nucleotide sequence shown by (1-1076) to 1369 in SEQ ID NO: 35 means that the sequence can start from position 1 in SEQ ID NO: 35 to The base of any of position 1076 terminates at the 1369th base of SEQ ID NO: 1. Promoter and its directed gene expression
  • an isolated nucleic acid having SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:
  • nucleic acid as shown in SEQ ID NO: 17 or a fragment of these sequences and a nucleotide sequence having a longer length comprising the sequence, the nucleic acid being useful as a promoter element for directing expression of a gene of interest.
  • the present invention also includes some variants of the above nucleic acids having the same function. include:
  • sequence is capable of interacting with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: under stringent conditions:
  • SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17
  • SEQ ID NO: 17 The nucleotide sequence shown in SEQ ID NO: 17 or a fragment thereof or an extended sequence which hybridizes and has a nucleic acid which directs a high level of expression of the gene of interest;
  • SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO:
  • nucleic acid having a nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or a fragment or an extended sequence thereof having 80% or more homology and having a high level of expression function indicative of a gene of interest ; or
  • SEQ ID NO: 2 SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: A nucleic acid of the indicated nucleotide sequence or a fragment thereof or an extended sequence that is complementary (preferably fully complementary).
  • Hybridization of polynucleotides is a technique well known to those skilled in the art, and the hybridization characteristics of a particular pair of nucleic acids indicate their similarity or identity. Accordingly, the present invention also relates to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ
  • the nucleotide sequence shown by ID NO: 17 or a fragment or extension thereof hybridizes and has at least 50%, preferably at least 70%, more preferably at least 80% (e.g., 85%, 90% 95) between the two sequences. % 96%, 97%, 98% or 99%) Nucleic acids of identity.
  • the invention particularly relates to polynucleotides that hybridize to the nucleic acids of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1 SDS, 60 °C ; or (2) hybridization a denaturant such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) only at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%.
  • the hybridizable nucleic acid also has a function of directing high level expression of the gene of interest.
  • the GUS gene or the GFP gene can be expressed at a high level under the guidance of the promoter of the present invention. Therefore, it can be seen that the promoter of the present invention is a promoter which is particularly suitable for guiding the expression of a gene of interest, and has important application value in the research of gene expression.
  • the promoter of the present invention can be operably linked to a gene of interest which can be foreign (heterologous) relative to the promoter.
  • the gene of interest may generally be any nucleic acid sequence (e.g., a structural nucleic acid sequence), and the gene of interest preferably encodes a protein having a particular function, such as certain proteins having important properties or functions.
  • the gene of interest includes, but is not limited to, a GUS gene, a GFP gene, a luciferase gene, and the like.
  • GUS is a good indicator of gene expression guided by the promoter. While the promoter of the present invention can direct the expression of GUS high expression, it is apparent that the promoter of the present invention as a gene regulatory element can direct the high expression of any other suitable gene.
  • the gene of interest may be a gene lacking or insufficiently expressed in a certain plant or a specific plant tissue or organ
  • the gene of interest and the promoter of the present invention may be operably Linking, or operably linking the gene of interest to the promoter of the present invention into a suitable vector, introducing it into an appropriate cell in an appropriate manner, and delivering it to the plant or a specific plant tissue or organ, thereby driving the gene of interest.
  • High water Flat expression may be a gene lacking or insufficiently expressed in a certain plant or a specific plant tissue or organ, and the gene of interest and the promoter of the present invention may be operably Linking, or operably linking the gene of interest to the promoter of the present invention into a suitable vector, introducing it into an appropriate cell in an appropriate manner, and delivering it to the plant or a specific plant tissue or organ, thereby driving the gene of interest.
  • High water Flat expression is a gene lacking or insufficiently expressed in a certain plant or a specific plant tissue or organ
  • the "plant” is not particularly limited as long as the plant is suitable for a genetic transformation operation (transgenic operation) such as various crops, flower plants, or forestry plants.
  • the plant may be, for example, a dicot, a monocot, or a gymnosperm.
  • cruciferous such as Chinese cabbage, Chinese cabbage
  • cruciferous such as Arabidopsis (such as Arabidopsis)
  • Gramineae such as rice
  • Solanaceae such as Tobacco
  • the plants include, but are not limited to: wheat, barley, rye, rice, corn, sorghum, beets, apples, pears, plums, peaches, apricots, cherries, strawberries, raspberries, blackberries, beans , lentils, peas, soybeans, rapeseed, mustard, poppy, olean, sunflower, coconut, castor oil plant, cocoa beans, peanuts, gourd, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, grape Pomelo, spinach, chicory, asparagus, cabbage, Chinese cabbage, pakchoi, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugar cane, tea, pepper, vine , ramie, bananas, natural rubber trees and ornamental plants.
  • the promoter of the present invention may also be operably linked to a modified gene sequence of interest which is exogenous (heterologous) relative to the promoter.
  • the gene of interest can be modified to produce a variety of desirable properties.
  • the gene of interest can be modified to increase the amount of essential amino acids, increase translation of amino acid sequences, alter post-translational modifications (such as phosphorylation sites), transport translation products out of the cell, improve protein stability, insert or delete Cell signal, etc.
  • promoters and genes of interest can be designed to downregulate specific genes. This is typically accomplished by ligating the promoter to the sequence of the gene of interest, which sequence is directed in antisense orientation. Those of ordinary skill in the art are familiar with such antisense techniques. Any nucleic acid sequence can be modulated in this manner.
  • the promoter and gene of interest sequences of the invention can be included in a recombinant vector.
  • the recombinant vector generally includes (from the 5' to 3' direction): a promoter that directs transcription of the gene of interest, and a gene of interest. If desired, the recombinant vector may also include a 3' transcriptional terminator, a 3' polynucleotideization signal, other non-translated nucleic acid sequences, a transport and targeting nucleic acid sequence, a resistance selection marker, an enhancer or an operator.
  • the recombinant vector comprises a promoter of the invention comprising a cloning site or at least one cleavage site downstream of the promoter.
  • the gene of interest is ligated into a suitable polycloning site or restriction site to operably link the gene of interest to the promoter.
  • expression vector refers to a bacterial plasmid, bacteriophage, yeast plasmid, virus or other vector well known in the art.
  • any plasmid and vector can be employed as long as it is capable of replication and stabilization in the host.
  • the expression vector is an eukaryotic expression vector.
  • expression vectors containing the promoters and/or gene sequences of interest described herein. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green Fluorescent protein
  • the recombinant vector may contain one or more other promoters in addition to the promoter of the present invention.
  • Such other promoters are, for example, tissue-specific, constitutive or inducible.
  • mannose synthase broccoli mosaic virus 19S and 35S CaMV19S CaMV35S
  • enhanced CaMV tobacco RB7 and the like.
  • the vector is a pCambia series vector. That is, using the multiple cloning site on pCambia, the promoter region of the present invention can be constructed in front of a reporter gene such as GUS or GFP, and the host cell can be transformed, and the promoter activates expression of a GUS or GFP-encoding gene, and the promoter is activated by a promoter. The regulation of each cis-acting element in the region mimics the state in which the gene is activated and transcribed in vivo.
  • a vector comprising the appropriate promoter and gene of interest described above can be used to transform a suitable host cell to enable expression of the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell.
  • Representative examples are: Escherichia coli, yeast, animal tissue cells, plant cells, and the like. It will be apparent to one of ordinary skill in the art how to select an appropriate vector and host cell.
  • an enhancer sequence is inserted into the vector.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, used as a promoter to enhance transcription of a gene.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 . Conversion can also be carried out by electroporation if desired.
  • the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, immature embryo transformation method, flower bud soaking method and the like.
  • Agrobacterium transformation or gene gun transformation such as leaf disc method, immature embryo transformation method, flower bud soaking method and the like.
  • plants can be regenerated by conventional methods to obtain transgenic plants.
  • a method for preparing a transgenic plant is: transferring a binary vector carrying a promoter and a gene of interest (both operably linked) into Agrobacterium, and Agrobacterium further integrates a vector fragment containing a promoter and a gene of interest Go to the chromosome of the plant.
  • the transgenic receptor plants involved are, for example, Arabidopsis thaliana, tobacco and rice.
  • a promoter which can direct high-level expression of a gene of interest is revealed, and a large amount of expression of a gene of interest can be efficiently initiated by the promoter of the present invention.
  • the present invention is a phenomenon in which the expression of a foreign gene leads to the silencing of a related gene and the introduction of rice during the breeding process.
  • Source promoters such as Cauliflower Mosaic Virus 35S Promoter (CaMV 35S) provide a new solution to the potential biosafety risks.
  • CaMV 35S Cauliflower Mosaic Virus 35S Promoter
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experimental Guide, Third Edition, Science Press, 2002, or according to the manufacturer. The suggested conditions. Percentages and parts are by weight unless otherwise stated.
  • the 5'-end regulatory region promoter of the Arabidopsis thaliana histone variant H3.3 gene HrR4 (AT4G40030) and the rice histone genes HrR7 (LOC_Os03g27310) and HTT ⁇ OC-OsC ⁇ gCMC O) were cloned.
  • the Arabidopsis gene H7 W promoter sequence is SEQ ID NO: 2; the rice gene HrR7 promoter sequence is SEQ ID NO: 3; the rice gene HTR712 promoter sequence is SEQ ID NO: 4; the Arabidopsis gene HTR1 promoter sequence As SEQ ID NO: 1, as a control.
  • Example 2 Arabidopsis thaliana gene HrR4 promoter drives the activity assay of GUS gene expression
  • the plant expression vectors pHTRl-GUS, pHTR4-GUS and CaMV35S-GUS were constructed using GUS as a reporter gene driven by the Arabidopsis thaliana HTI and HTR4 promoters and CaMV35S.
  • the Arabidopsis thaliana gene HTR1, promoter, and CaMV35S sequences were inserted into the Hindlll and BamHI restriction sites of the pCambial300 expression vector (purchased from Cambia) (i.e., upstream of the GUS reporter gene).
  • the GUS element was inserted into the B mH/he Sac/cleavage site of pCambial300.
  • the recombinant vectors obtained were designated as: pCambial 300-pHTRl-GUS, pCambial 300-pHTR4-GUS and pCambial300-CaMV35S-GUS.
  • the constructed recombinant vector was transformed into Agrobacterium EH4105 (see Hood, E. E. et al., Transgenic Res., 1993, 2, 208-218), and positive clones were screened.
  • the obtained Agrobacterium containing the plasmid of the target gene was transfected into tobacco leaves, and after cocultivation for 2 days, it was transferred to a screening medium to remove residual Agrobacterium.
  • the positive callus infected with Agrobacterium was histochemically stained for about one month to detect the transient expression of the GUS gene.
  • the buds are cut into the rooting medium containing the resistance, and the rooted tobacco seedlings are transferred to the pots in about one month, and cultured in the greenhouse, and the leaves are taken in about 1 month. Histochemical staining was performed to detect the transient expression of the Gt/S gene.
  • the co-cultured callus or leaves are rinsed with sterile distilled water and placed in a 1.5 ml centrifuge tube.
  • the GUS staining solution of the wound tissue was mixed and the callus was fully contacted with the staining solution; the bath was incubated at 37 ° C for 2 hours or overnight; the callus was decolorized with 70% ethanol as a decolorizing solution and blue spots were observed.
  • HrR4 The promoter of HrR4 (H3.3) is highly expressed in the intercellular phase and is constitutively expressed at high levels in vegetative organs.
  • the expression of HTR1 (H3.1) is closely related to DNA replication, and a large number of S phase H3.1 is synthesized during DNA replication.
  • the results of GUS histochemical staining indicated that the expression of Gt/S gene driven by HTR1 promoter in Arabidopsis thaliana was significantly higher than that of HTR4 and CaMV35S-driven Gt/S expression, as shown in Figure 1.
  • the expression activity of the Gt/S gene driven by the H7 W promoter of Arabidopsis thaliana gene was significantly higher than that of HTR1, as shown in Figure 2.
  • the Arabidopsis gene HTR1 and H7 W promoters were more active than the commonly used constitutive promoter CaMV35S.
  • the constructed plant expression vectors pCambial300-pHTRl-GUS, P Cambial300-pHTR4-GUS and P Cambial300-CaMV35S-GUS were transformed into Agrobacterium GV W (Invitrogen), and positive clones were selected.
  • the obtained Agrobacterium containing the plasmid of the target gene was transfected into the Arabidopsis inflorescence, and the Arabidopsis thaliana seedlings were wrapped in plastic wrap for 2 days, then transferred to light culture, and the seeds were collected after about one and a half months in the greenhouse culture.
  • the TO seeds were screened on the screening medium, and the T1 generation seedlings were identified and transferred into pots. After 2 weeks of greenhouse culture, the leaves were subjected to histochemical staining to detect the transient expression of Gt/S gene.
  • GyJiOi Invitrogen, screening positive clones.
  • the obtained Agrobacterium containing the plasmid of the target gene was transfected into the Arabidopsis thaliana, and the Arabidopsis thaliana seedlings were wrapped in plastic wrap for 2 days, then transferred to light culture, and the seeds were collected after about one and a half months in the greenhouse.
  • the T0 generation seeds were screened on the screening medium, and the T1 generation seedlings were identified and transferred to the pots. After 2 weeks of culture in the greenhouse, the leaves were extracted for RNA and reverse transcribed into cDNA for quantitative PCR reaction.
  • HTR1 and H7 W promoters endogenous can be detected by detecting the cDNA of HTR1 and HTR4 genes. Expression reflects that since Copl is an endogenous gene of Arabidopsis thaliana, the expression level of the CaMV35S promoter is reflected by detecting the activity of YFP.
  • RT-PCR detection primers are as follows:
  • HTR1 F: CGTACCAAGCAAACCGCAAGGAAA (SEQ ID NO: 7);
  • HTR4-. F TGCACCAACTACTGGTGGAGTCAA (SEQ ID NO: 9);
  • R AGCTAAGACAGCATGGCTCTGGAA (SEQ ID NO: 10);
  • YFP F: TGACCCTGAAGTTCATCTGCACCA (SEQ ID NO: 11);
  • R TCTTGTAGTTGCCGTCGTCCTTGA (SEQ ID NO: 12).
  • Figure 4 shows the difference maps of the Ct values of the HTR1, HTR4, and YFP genes.
  • the inventors selected three transgenic Arabidopsis thaliana seedlings transfected with pCambial301-CaMV35S-Copl-YFP as the material, and the abscissa was 3 samples (35S-1). , 35S-2, 35S-3), the ordinate is the Ct value.
  • Ht W gene had the smallest Ct value and HTR1 was slightly lower than 1 ⁇ . This reflects the strongest expression of the H7 W promoter in the leaves of three transgenic Arabidopsis thaliana.
  • Example 3 Rice gene HTR 711 and HTR 712 promoter drive GUS gene expression
  • the plant expression vectors pCambial300-pHTR711-GUS, pCambial300-pHTR712-GUS and P Cambial300-CaMV35S-GUS were constructed with GUS as a reporter gene driven by the rice gene HTR711 and HTR712 promoters.
  • the constructed plasmid was transformed into Agrobacterium EH4105 and screened. Positive clone.
  • the rice gene HTR711 and HTR712 promoters, CaMV35S sequences were inserted into the ⁇ / /// and BamHI restriction sites of the pCambial300 expression vector (ie, upstream of the GUS reporter gene).
  • the GUS element was inserted into the BamH/Whit SotJ restriction site of pCambial300.
  • the obtained Agrobacterium containing the plasmid of interest was transfected into the calli of rice transforming receptor (Zhonghua 11), and after co-cultivation for two days, it was transferred to a screening medium to remove residual Agrobacterium; after three days, it was infected with Agrobacterium positive.
  • the callus was subjected to histochemical staining to detect the transient expression of the GUS gene.
  • the co-cultured callus was rinsed with sterile distilled water, placed in a 1.5 ml centrifuge tube, and the GUS staining solution without callus was added and mixed to make the callus fully contact with the staining solution; warm bath at 37 °C 2 hours or overnight; callus was decolorized with 70% ethanol as a decolorizing solution and blue spots were observed.
  • Example 4 Rice gene HTR 711 and HTR 712 promoter drive GFP gene transient expression analysis Driven by rice gene HTR711 and HTR712 promoter, green fluorescent protein (GFP) is used as a reporter gene. Plant expression vectors pCambial300-pHTR711-GFP and pCambial300-pHTR712-GFP were constructed.
  • the rice gene HTR711 and HTR712 promoters and the CaMV35S sequence were inserted into the Hindlll and BamHI restriction sites of the pCambial300 expression vector (i.e., upstream of the GFP gene).
  • the GFP element was inserted into the BamH/Whit SotJ restriction site of pCambial300.
  • the constructed plasmid was transformed into Agrobacterium EH4105 and positive clones were screened.
  • the three-leaf stage foxtail Green foxtail, Setaria viridis (L.) P. Beauv) was selected for transient expression of the receptor material.
  • Monoclones picking pCambial300-pHTR711-GFP and pCambial300-pHTR712-GFP positive strain EH4105 were cultured at 28 ° C, 200 rpm until the OD 6TO was about 0.8.
  • pCambial300-pHTR711-GFP and pCambial300-pHTR712-GFP plasmid in foxtail leaves was observed by deconvolutance fluorescence microscopy.
  • the leaves of the size of 1 ⁇ 2 cm 2 in the injection area were placed on a glass slide with a drop of distilled water, and the expression of GFP was observed with a water microscope lens with a magnification of 60 times.
  • pHTR4 and pHTR711 7 fragments of different lengths were taken to identify their key sites.
  • the Arabidopsis thaliana and rice genomes were used as templates, and the primers in Table 1 were used.
  • the reverse primers were reverse PHTR4-R-BamHI;
  • the reverse primers are all reverse PHTR711-R-BamHI) to amplify the fragment, and the yellow fluorescent protein (YFP) is used as a plant expression vector for the reporter gene. Seven vectors were constructed, and the primer sequences are shown in Table 1.
  • the rice gene HTR711 promoter was inserted into the pCambial300 expression vector Hindlll and BamHI restriction sites (i.e., upstream of the YFP gene), and the YFP element was inserted into the pCambial300 and the SotJ restriction site.
  • the constructed plasmids were separately transformed into Agrobacterium GV, and positive clones were screened.
  • Tobacco SAW was selected as a transient expression receptor material.
  • the monoclonal clone carrying the positive strain GV3101 was cultured at 28 ° C, 200 rpm until the OD 6TO was about 0.8.
  • the key segments of the two genes HTR4 and HTR711 promoters can be derived from the fluorescence values in Figure 9-10 and the box plot in Figure 11. From the experimental results, it can be seen that the key region of the Arabidopsis thaliana H7 W promoter is located at 453-663 bp from the translation initiation site, and the key region of the rice gene HTR711 promoter is located 201-321 bp from the translation initiation site. Because the Arabidopsis gene HTR4 promoter has a 463 bp intron between the translation initiation site and the transcription initiation site, the key regions of both promoters are less than 100 bp from the transcription start site. position.
  • Example 6 Transgenic Arabidopsis thaliana GUS histochemical staining assay for promoter activity
  • the plant expression vectors pCambial300-pHTRl-GUS, P Cambial300-pHTR4-GUS and pCambial300-CaMV35S-GUS constructed as described above were transformed into Agrobacterium GV W (Invitrogen), and positive clones were selected.
  • Agrobacterium GV W Invitrogen
  • the Agrobacterium containing the plasmid of the target gene was transfected into the Arabidopsis inflorescence, and the Arabidopsis thaliana seedlings were wrapped in plastic wrap for 2 days, then transferred to light culture, and the seeds were collected after about one and a half months in the greenhouse.
  • the TO seeds were screened on the screening medium, and the T1 generation seedlings were identified and transferred to the pots. After the greenhouse culture was collected, the collected seeds were placed in the screening medium. After about 10 days, 30 whole plants were taken for tissue culture. Chemical staining was used to detect the transient expression of the Gt/S gene.
  • Gt/ S is a reporter gene constructing plant expression vectors pHTR101-GUS, pHTR102-GUS, pHTR104-GUS and pHTR113-GUS.
  • the promoter sequences were inserted into the ⁇ / / / / and BamH / restriction sites of the pCambial300 expression vector (purchased from Cambia) (i.e., upstream of the GUS reporter gene).
  • the GUS element was inserted into the B mH/ and SotJ cleavage site of pCambial300.
  • the obtained recombinant vectors were named as: pCambial300-pHTR101-GUS, pCambial 300-pHTR102-GUS, pCambial300-pHTR104-GUS and pCambial300-pHTR113-GUS.
  • the plant expression vectors pCambial300-pHTR101-GUS, pCambial300-pHTR102-GUS, pCambial 300-pHTR104-GUS and pCambial300-pHTR113-GUS were transformed into Agrobacterium tumefaciens GyJiOi (Invitrogen), and positive clones were selected.
  • the obtained Agrobacterium containing the plasmid of the target gene was transfected into the Arabidopsis thaliana, and the Arabidopsis thaliana seedlings were wrapped in plastic wrap for 2 days, then transferred to light culture, and the seeds were collected after about one and a half months in the greenhouse.
  • the T0 generation seeds were screened on the screening medium, and the T1 generation seedlings were identified and transferred to the pots. After 2 weeks of greenhouse culture, the leaves were subjected to histochemical staining to detect the transient expression of the Gt/S gene, as shown in Fig. 13.
  • the results indicate that the maize H3.3 gene HTR101 (SEQ ID NO: 13), HTR102 (SEQ ID NO: 14), HTR104 (SEQ ID NO: 15), and HrR _? (SEQ ID NO: 16) promoters are all driven.
  • HTR10 SEQ ID NO: 15 HTR101 (SEQ ID NO: 13), HTR102 (SEQ ID NO: 14), HTR113 (SEQ ID NO: 16) .
  • Example 8 Transgenic Arabidopsis thaliana GUS histochemical staining for detection of rice promoter activity
  • the plant expression vector pHTR706-GUS was constructed using Gt/S as a reporter gene driven by the rice ⁇ 3.1 gene HrR70 ⁇ promoter (SEQ ID NO: 17).
  • the rice gene HTR 70 (promoter sequence was inserted into the Hindlll and BrahHI restriction sites of the pCambia 1300 expression vector (purchased from Cambia), respectively (ie located upstream of the GUS reporter gene).
  • the GUS element was inserted into the BamHI of P Cambial 300.
  • Sacl cleavage site The obtained recombinant vector was named: pCambial 300-pHTR706-GUS.
  • the constructed plant expression vector pCambial 300-pHTR706-GUS was transformed into Agrobacterium GyJiOi (Invitrogen), and positive clones were selected.
  • the obtained Agrobacterium containing the plasmid of the target gene was transfected into the Arabidopsis inflorescence, and the Arabidopsis thaliana seedlings were wrapped in plastic wrap for 2 days, then transferred to light culture, and the seeds were collected after about one and a half months in the greenhouse culture.
  • the TO seeds were screened on the screening medium, and the T1 generation seedlings were identified and transferred to the pots.
  • the transgenic Arabidopsis thaliana pCambial 300-pHTR706-GUS was cultured for 2 weeks in the greenhouse, and the leaves were subjected to histochemical staining to detect the Gt/S gene. Transient expression, as shown in Figure 14.

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Abstract

Provided is a promoter having high-efficiency initiating activity that is derived from the polynucleotide of the gene of plant histone variant 3.3. The promoter has a wide range of activities and is capable of guiding high-level expression of a target gene.

Description

一种具有高效驱动活性的启动子  A promoter with efficient driving activity
技术领域  Technical field
本发明属于生物技术和植物学领域； 更具体地， 本发明涉及一种具有高效驱动活性的 启动子。 背景技术  The present invention is in the field of biotechnology and botany; more specifically, the present invention relates to a promoter having efficient driving activity. Background technique
在植物基因工程中常用的启动子按其作用和功能分为三种： 组成型启动子、 诱导型 启动子和组织特异型启动子。 其中组成型启动子最为常用。 组成型启动子分为内源组成 型和异源组成型启动子两大类。 植物基因工程中常用的内源启动子主要有水稻肌动蛋白 (actin)基因和玉米泛素 (ubiquitin)基因的启动子。 异源组成型启动子有来自农杆菌的 Nos 和 Ocs启动子以及来源于烟草花叶病毒基因的 CaMV 35S启动子。 组成型启动子在所有 组织中都有表达， 具有持续性但不具有时空特异性。 重复使用同一启动子驱动两个或两 个以上的外源基因可能引起基因沉默或共抑制现象。 然而关于 CaMV35S启动子在转基 因生物安全性角度存在潜在风险的问题报道比较多。 已有文献报道认为 CaMV35S启动 子来源于烟草花叶病毒， 一旦在转基因过程中整合到病毒隐性基因附近就有可能激活病 毒的表达， 或者 CaMV35S启动子***到编码毒素蛋白的基因上游， 将增强毒素蛋白的 表达， 使得转基因植物的安全性面临巨大的挑战。  Promoters commonly used in plant genetic engineering are classified into three types according to their functions and functions: constitutive promoters, inducible promoters, and tissue-specific promoters. Among them, constitutive promoters are most commonly used. The constitutive promoter is divided into two major categories: endogenous constitutive and heterologous constitutive promoter. Endogenous promoters commonly used in plant genetic engineering mainly include the promoters of the rice actin gene and the ubiquitin gene. Heterologous constitutive promoters include the Nos and Ocs promoters from Agrobacterium and the CaMV 35S promoter derived from the Tobacco Mosaic Virus gene. The constitutive promoter is expressed in all tissues and is persistent but not spatiotemporal specific. Repetitive use of the same promoter to drive two or more foreign genes may cause gene silencing or cosuppression. However, there are many reports on the potential risks of the CaMV35S promoter in terms of biosafety of transgenic genes. It has been reported in the literature that the CaMV35S promoter is derived from tobacco mosaic virus, and it may activate the expression of the virus once it is integrated into the recessive gene in the transgene process, or the CaMV35S promoter is inserted upstream of the gene encoding the toxin protein. The expression of toxin proteins poses a huge challenge to the safety of transgenic plants.
真核生物的细胞核中与 DNA 结合的蛋白质包括组蛋白和非组蛋白类。 组蛋白指细 胞核中与 DNA结合的碱性蛋白质的总称。 组蛋白富含精氨酸和赖氨酸等碱性氨基酸， 二 者加起来约为所有氨基酸残基的 1/4。 它们分子量较小， 大约 10 000〜20 000道尔顿。 组蛋白主要有五种—— Hl、 H2A、 H2B、 H3、 H4， 它们具有不同的分子量和氨基酸组成。 在五种组蛋白中， 四种组蛋白 (H2A、 H2B、 H3、 H4) 分子量较小（102〜135个氨基酸残 基)， HI的分子量较大 (约含 220个氨基酸残基)， 序列保守程度相对较低， 但结构相似， HI组蛋白其球形中心结构域在进化上保守， 而 N端和 C端两个 "臂" 的氨基酸变异较 大， 所以 HI在进化上不如核小体组蛋白那么保守， 在构成核小体时 HI起连接作用， 它赋予染色质以极性。  Proteins that bind to DNA in the nucleus of eukaryotes include histones and non-histones. Histone refers to a general term for basic proteins that bind to DNA in the nucleus. Histones are rich in basic amino acids such as arginine and lysine, which add up to about 1/4 of all amino acid residues. They have a small molecular weight of about 10 000 to 20 000 Daltons. There are five major histones - Hl, H2A, H2B, H3, H4, which have different molecular weights and amino acid compositions. Among the five histones, the four histones (H2A, H2B, H3, H4) have a small molecular weight (102 to 135 amino acid residues), and the molecular weight of HI is large (about 220 amino acid residues), and the sequence is conservative. The degree is relatively low, but the structure is similar. The spherical central domain of HI histone is evolutionarily conserved, while the amino acids of the two "arms" at the N-terminus and C-terminus are larger, so HI is not as evolutionary as nucleosomal histone. So conservative, HI acts as a linker when it forms a nucleosome, which imparts polarity to chromatin.
除常规的组蛋白外， 组蛋白还有各种不同的变体， 尤其是在核心组蛋白 H2A、 H3 和连接组蛋白 HI中。 核心组蛋白 H2B和 H4变化相对较少。 根据与对应的常规组蛋白 氨基酸序列的不同， 组蛋白变体可分为两类： 一类是由氨基酸替换产生的变体， 氨基酸 总数不变， 称为同型变体 (Homomorphous variants), 如植物常规组蛋白 H3(即 H3.1)及其 变体 H3.3。 它们之间只在四个位置氨基酸呈现不同， 分别在第 31位 (H3.1中为甘氨酸， H3.3中为苏氨酸）、 41位 (H3.1中为苯丙氨酸， H3.3中为酪氨酸）、 87位 (H3.1中为半胱 氨酸， H3.3中为组氨酸)和 90位 (H3.1中为甘氨酸， H3.3中为亮氨酸)。虽然 H3.1和 H3.3 之间只有四个氨基酸的区别， 但它们在染色质上的分布和功能有很大不同。 H3.1主要在 DNA复制过程中加载入染色质， 而 H3.3则可以不依赖于 DNA复制而加载入间期染色 质。 H3.1主要与沉默的染色质有关， 而 H3.3主要与转录活跃的染色质有关。 在植物包 括拟南芥、 水稻和玉米组蛋白 H3.3的氨基酸序列分析结果中发现植物的组蛋白 H3.3非 常保守。 In addition to conventional histones, histones also have a variety of variants, particularly in the core histones H2A, H3 and linked histone HI. The core histones H2B and H4 vary relatively little. Histone variants can be divided into two categories depending on the amino acid sequence of the corresponding conventional histones: one is a variant produced by amino acid substitution, and the total number of amino acids is unchanged, called Homomorphous variants, such as plants. Conventional histone H3 (ie H3.1) and its variant H3.3. There are only four amino acids in between, which are in the 31st position (glycine in H3.1, threonine in H3.3) and 41 (phenylalanine in H3.1, H3. 3 is tyrosine), 87 (cysteine in H3.1, histidine in H3.3) and 90 (glycine in H3.1, leucine in H3.3) . Although there are only four amino acid differences between H3.1 and H3.3, their distribution and function in chromatin are quite different. H3.1 is mainly loaded into chromatin during DNA replication, while H3.3 can be loaded into interphase staining independent of DNA replication. Quality. H3.1 is primarily associated with silent chromatin, whereas H3.3 is primarily involved in transcriptionally active chromatin. The histone H3.3 of the plants was found to be very conserved in the results of amino acid sequence analysis of plants including Arabidopsis thaliana, rice and maize histone H3.3.
因此， 结合现有的植物工程技术， 对高表达保守蛋白的启动子的功能研究是非常必 要的。 发明内容  Therefore, in combination with existing plant engineering techniques, functional studies of promoters that highly express conserved proteins are essential. Summary of the invention
本发明的目的在于提供一种具有高效驱动活性的启动子及其应用。  It is an object of the present invention to provide a promoter having efficient driving activity and an application thereof.
在本发明的第一方面， 提供分离的多核苷酸， 所述的多核苷酸是：  In a first aspect of the invention, an isolated polynucleotide is provided, said polynucleotide being:
(1) SEQ ID NO: 2所示的核苷酸序列的多核苷酸；  (1) a polynucleotide of the nucleotide sequence shown by SEQ ID NO: 2;
(2) SEQ ID NO: 3所示的核苷酸序列的多核苷酸；  (2) a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 3;
(3) SEQ ID NO: 4所示的核苷酸序列的多核苷酸；  (3) a polynucleotide of the nucleotide sequence shown by SEQ ID NO: 4;
(4) SEQ ID NO: 34中（1-743)〜 1405位所示的核苷酸序列的多核苷酸；  (4) a polynucleotide of the nucleotide sequence shown by (1-743) to 1405 in SEQ ID NO: 34;
(5) SEQ ID NO: 35中（1-1076)〜1369位所示的核苷酸序列的多核苷酸； (6) SEQ ID NO: 13所示的核苷酸序列的多核苷酸；  (5) a polynucleotide of the nucleotide sequence shown by (1-1076) to 1369 in SEQ ID NO: 35; (6) a polynucleotide of the nucleotide sequence shown by SEQ ID NO: 13;
(7) SEQ ID NO: 14所示的核苷酸序列的多核苷酸；  (7) a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 14;
(8) SEQ ID NO: 15所示的核苷酸序列的多核苷酸；  (8) a polynucleotide of the nucleotide sequence shown by SEQ ID NO: 15;
(9) SEQ ID NO: 16所示的核苷酸序列的多核苷酸；  (9) a polynucleotide of the nucleotide sequence shown by SEQ ID NO: 16;
(10) SEQ ID NO: 17所示的核苷酸序列的多核苷酸；  (10) a polynucleotide of the nucleotide sequence shown by SEQ ID NO: 17;
(11) 核苷酸序列在严格条件下能够与 (1)-(10)任一限定的多核苷酸序列杂交且具有 (11) The nucleotide sequence is capable of hybridizing under stringent conditions to any of the polynucleotide sequences defined in (1) to (10) and has
(1)-(10)任一限定的多核苷酸功能的多核苷酸； 或 (1)-(10) any polynucleotide having a defined polynucleotide function; or
(12) 核苷酸序列与 (1)-(10)任一限定的多核苷酸序列有 80%以上 (较佳地 85%以上； 更佳地 90%以上； 更佳地 95%以上； 更佳地 99%以上)相同性且具有（1)-(10)任一限定的 多核苷酸功能的多核苷酸。  (12) The nucleotide sequence is 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more; more preferably) of the polynucleotide sequence defined by any one of (1) to (10) Preferably, 99% or more of the polynucleotides having the same identity and having the polynucleotide function defined by any of (1) to (10).
在另一优选例中， （4)中， 包括： SEQ ID NO: 34中第 464-1405位 (pHTR4-5(942bp)) 所示的核苷酸序列的多核苷酸； SEQ ID NO: 34中第 276-1405位 (pHTR4-6(1130bp))所示 的核苷酸序列的多核苷酸； 或 SEQ ID NO: 34所示的核苷酸序列的多核苷酸。  In another preferred embodiment, (4), comprising: a polynucleotide of the nucleotide sequence shown in position 464-1405 (pHTR4-5 (942 bp)) of SEQ ID NO: 34; SEQ ID NO: 34 A polynucleotide of a nucleotide sequence shown in position 276-1405 (pHTR4-6 (1130 bp)); or a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 34.
在另一优选例中， （5)中， 包括： SEQ ID NO: 3中第 201-521位 (pHTR711-2(521bp)) 所示的核苷酸序列的多核苷酸； SEQ ID NO: 35中第 624-1369位 (pHTR711-4(746bp))所 示的核苷酸序列的多核苷酸； SEQ ID NO: 35中第 416-1369位 (pHTR711-5(954bp))所示 的核苷酸序列的多核苷酸； SEQ ID NO: 35中第 200-1369位 (pHTR711-6(1170bp))所示的 核苷酸序列的多核苷酸； 或 SEQ ID NO: 35所示的核苷酸序列的多核苷酸。  In another preferred embodiment, (5), comprising: a polynucleotide of the nucleotide sequence shown at positions 201-521 (pHTR711-2 (521 bp)) in SEQ ID NO: 3; SEQ ID NO: 35 a polynucleotide of a nucleotide sequence shown in position 624-1369 (pHTR711-4 (746 bp)); a nucleoside represented by position 416-1369 (pHTR711-5 (954 bp)) in SEQ ID NO: 35 a polynucleotide of an acid sequence; a polynucleotide of a nucleotide sequence shown at position 200-1369 (pHTR711-6 (1170 bp)) in SEQ ID NO: 35; or a nucleotide represented by SEQ ID NO: Sequence polynucleotide.
在本发明的另一方面， 提供所述的多核苷酸的用途， 用于作为启动子元件。  In another aspect of the invention, the use of the polynucleotide is provided for use as a promoter element.
在一个优选例中， 所述的启动子元件用于指导目的基因强表达。  In a preferred embodiment, the promoter element is used to direct strong expression of the gene of interest.
在另一优选例中， 所述的目的基因是结构基因。 在另一优选例中， 所述的目的基因可编码具有特定功能的蛋白。 In another preferred embodiment, the gene of interest is a structural gene. In another preferred embodiment, the gene of interest encodes a protein having a specific function.
在另一优选例中， 所述的目的基因是外源基因。  In another preferred embodiment, the gene of interest is a foreign gene.
在本发明的另一方面， 提供一种载体， 所述的载体含有所述的多核苷酸， 作为启动 子元件。  In another aspect of the invention, a vector is provided, the vector comprising the polynucleotide as a promoter element.
在一个优选例中， 所述的载体还含有与所述的多核苷酸操作性连接的目的基因。 在另一优选例中， 所述的目的基因位于所述多核苷酸的下游。  In a preferred embodiment, the vector further comprises a gene of interest operably linked to the polynucleotide. In another preferred embodiment, the gene of interest is located downstream of the polynucleotide.
在另一优选例中， 所述的目的基因位于所述多核苷酸的下游， 且与所述多核苷酸的 间隔小于 lOOObp; 优选的， 小于 500bp ; 更优选的， 小于 200bp。  In another preferred embodiment, the gene of interest is located downstream of the polynucleotide and is less than 1000 bp from the polynucleotide; preferably, less than 500 bp; more preferably, less than 200 bp.
在另一优选例中， 所述的目的基因位于所述多核苷酸的下游， 且与所述多核苷酸的 间隔小于 100bp， 更佳地小于 50bp。  In another preferred embodiment, the gene of interest is located downstream of the polynucleotide and is less than 100 bp, more preferably less than 50 bp from the polynucleotide.
在另一优选例中， 所述的载体是真核表达载体。  In another preferred embodiment, the vector is a eukaryotic expression vector.
在另一优选例中， 在所述多核苷酸的下游包含多克隆位点或至少一个酶切位点。 在本发明的另一方面， 提供一种遗传工程化的宿主细胞 (较佳地， 所述的宿主细胞不 是动植物繁殖材料)， 所述的细胞：  In another preferred embodiment, a multiple cloning site or at least one cleavage site is included downstream of the polynucleotide. In another aspect of the invention, a genetically engineered host cell is provided (preferably, said host cell is not an animal or plant propagation material), said cell:
含有所述的载体； 或其基因组中整合有外源的所述的多核苷酸。  Containing the vector; or a polynucleotide having the foreign gene integrated in its genome.
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的。 附图说明  Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS
图 1、 拟南芥基因 HTR1和 H7 W启动子驱动 Gt/S基因表达图。  Figure 1. Arabidopsis thaliana genes HTR1 and H7 W promoters drive Gt/S gene expression maps.
A: PCambial300-CaMV35S-GUS转化烟草，在愈伤组织中 GUS组织化学染色结果；A: P Cambial300-CaMV35S-GUS transformed tobacco, GUS histochemical staining results in callus;
B : pCambial300-pHTRl-GUS转化烟草， 在愈伤组织中 GUS组织化学染色结果； C: pCambial300-pHTR4-GUS转化烟草， 在愈伤组织中 GUS组织化学染色结果。 图 2、 拟南芥基因 HTR1和 H7 W启动子驱动 GUS基因表达图。 B: pCambial300-pHTRl-GUS transformed tobacco, GUS histochemical staining results in callus; C: pCambial300-pHTR4-GUS transformed tobacco, GUS histochemical staining results in callus. Figure 2. Arabidopsis gene HTR1 and H7 W promoters drive GUS gene expression.
A: pCambial300-pHTRl-GUS转化烟草， 在烟草叶片中 GUS组织化学染色结果； A: pCambial300-pHTRl-GUS transformed tobacco, GUS histochemical staining results in tobacco leaves;
B : pCambial300-pHTR4-GUS转化烟草， 在烟草叶片中 GUS组织化学染色结果； C: PCambial300-CaMV35S-GUS转化烟草，在烟草叶片中 GUS组织化学染色结果。 图 3、 拟南芥基因 HTR1和 H7 W启动子驱动 GUS基因表达图。 B: pCambial300-pHTR4-GUS transformed tobacco, GUS histochemical staining results in tobacco leaves; C: P Cambial300-CaMV35S-GUS transformed tobacco, GUS histochemical staining results in tobacco leaves. Figure 3. Arabidopsis gene HTR1 and H7 W promoters drive GUS gene expression maps.
Al , A2: pCambial300-pHTRl-GUS转化拟南芥， 在拟南芥莲座叶、 茎生叶和花序 中的 GUS组织化学染色结果；  Al, A2: pCambial300-pHTRl-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in Arabidopsis rosette leaves, stem leaves and inflorescences;
B l， B2: pCambial300-pHTR4-GUS转化拟南芥， 在拟南芥莲座叶、 茎生叶和花序 中 GUS组织化学染色结果；  B l, B2: pCambial300-pHTR4-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in Arabidopsis rosette leaves, stem leaves and inflorescences;
Cl， C2: pCambial300-CaMV35S-GUS转化拟南芥， 在拟南芥莲座叶、 茎生叶和花 序中 GUS组织化学染色结果。 图 4、 转基因拟南芥中 HTR1 , HTR4和 YFP的 Ct值差异图。 横坐标为三株样本， 分别是 35S-1 , 35S-2, 35S-3 ; 纵坐标为 Ct值。 图 5、 水稻基因 HTR711和 HTR712启动子驱动 Gt/S基因表达图。 Cl, C2: pCambial300-CaMV35S-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in Arabidopsis rosette leaves, stem leaves and inflorescences. Figure 4. Differences in Ct values for HTR1, HTR4 and YFP in transgenic Arabidopsis. The abscissa is three samples, which are 35S-1, 35S-2, 35S-3, respectively; the ordinate is the Ct value. Figure 5. Rice gene HTR711 and HTR712 promoters drive Gt/S gene expression maps.
A: pCambial300-CaMV35S-GUS转化中花 11愈伤组织， GUS组织化学染色结果； B : pCambial300-pHTR711-GUS转化中花 11愈伤组织， GUS组织化学染色结果； C: pCambial300-pHTR712-GUS转化中花 11愈伤组织， GUS组织化学染色结果。 图 6、 水稻基因 HTR711和 HrR7J2启动子驱动 GFP基因表达在亚细胞水平的定位 图。  A: pCambial300-CaMV35S-GUS transformed flower callus, GUS histochemical staining results; B: pCambial300-pHTR711-GUS transformed flower 11 callus, GUS histochemical staining results; C: pCambial300-pHTR712-GUS transformation Zhonghua 11 callus, GUS histochemical staining results. Figure 6. Mapping of rice gene HTR711 and HrR7J2 promoters driving GFP gene expression at the subcellular level.
A: 水稻基因 HTR711和 HTR712启动子驱动 GFP基因在三叶期狐尾草叶片细胞核 中的表达情况；  A: Rice gene HTR711 and HTR712 promoter drive the expression of GFP gene in the nucleus of Trifolium repens leaves;
B : 在单个狐尾草叶片细胞中 基因的表达情况；  B: gene expression in a single foxtail leaf cell;
C: 狐尾草植株（Green foxtail, ^ απ'ύί Wn'6¾(L.) P. Beauv)。 图 7、 7个拟南芥基因 H7 W启动子片段示意图。其中， pHTR4-l(116bp)代表 SEQ ID NO: 2序列中第 548-663位； pHTR4-2(263bp)代表 SEQ ID NO: 2序列中第 401-663位； pHTR4-3(453bp)代表 SEQ ID NO: 2序列中第 211-663位； pHTR4-4(663bp)即 SEQ ID NO: 2序列； pHTR4-5(942bp)序列如 SEQ ID NO: 34中第 464-1405位； pHTR4-6(1130bp)序 列如 SEQ ID NO: 34中第 276-1405位； pHTR4-7(1405bp)序列如 SEQ ID NO: 34序列。 图 8、 7个水稻基因 HTR711启动子片段示意图。其中， pHTR711-l(201bp)代表 SEQ ID NO: 3序列中第 321-521位； pHTR711-2(321bp)代表 SEQ ID NO: 3序列中第 201-521 位； pHTR711-3(521bp)代表 SEQ ID NO: 3序列； pHTR711-4(746bp)序列如 SEQ ID NO: 35 中第 624-1369 位； pHTR711-5(954bp)序列如 SEQ ID NO: 35 中第 416-1369 位； pHTR711-6(1170bp)序列如 SEQ ID NO: 35中第 200-1369位； pHTR711-7序列如 SEQ ID NO: 35序列。 图 9、 7个拟南芥基因 H7 W启动子的荧光图。  C: foxtail plant (Green foxtail, ^ απ'ύί Wn'63⁄4 (L.) P. Beauv). Figure 7. Schematic diagram of seven Arabidopsis thaliana H7 W promoter fragments. Wherein, pHTR4-l (116 bp) represents position 548-663 in the sequence of SEQ ID NO: 2; pHTR4-2 (263 bp) represents position 401-663 in the sequence of SEQ ID NO: 2; pHTR4-3 (453 bp) represents SEQ ID NO: position 21-263 in the 2 sequence; pHTR4-4 (663 bp) is the sequence of SEQ ID NO: 2; pHTR4-5 (942 bp) sequence is as position 464-1405 in SEQ ID NO: 34; pHTR4-6 ( The 1130 bp) sequence is located at positions 276-1405 of SEQ ID NO: 34; the pHTR4-7 (1405 bp) sequence is SEQ ID NO: 34 sequence. Figure 8. Schematic diagram of seven rice gene HTR711 promoter fragments. Wherein, pHTR711-1 (201 bp) represents position 321-521 of the sequence of SEQ ID NO: 3; pHTR711-2 (321 bp) represents position 201-521 of the sequence of SEQ ID NO: 3; pHTR711-3 (521 bp) represents SEQ ID NO: 3 sequence; pHTR711-4 (746 bp) sequence is located at positions 624-1369 of SEQ ID NO: 35; pHTR711-5 (954 bp) sequence is at positions 416-1369 of SEQ ID NO: 35; pHTR711-6 ( The 1170 bp) sequence is in positions 200-1369 of SEQ ID NO: 35; the pHTR711-7 sequence is in SEQ ID NO: 35 sequence. Figure 9. Fluorescence of seven Arabidopsis gene H7 W promoters.
A: pHTR4-l启动子的荧光图；  A: Fluorescence map of the pHTR4-l promoter;
B : pHTR4-2启动子的荧光图；  B: Fluorescence map of the pHTR4-2 promoter;
C: pHTR4-3启动子的荧光图；  C: Fluorescence map of the pHTR4-3 promoter;
D: pHTR4-4启动子的荧光图；  D: Fluorescence of the pHTR4-4 promoter;
E: pHTR4-5启动子的荧光图； F: pHTR4-6启动子的荧光图； E: Fluorescence of the pHTR4-5 promoter; F: Fluorescence map of the pHTR4-6 promoter;
G: pHTR4-7启动子的荧光图。 图 10、 7个水稻基因 HrR7J 启动子的荧光图。  G: Fluorescence of the pHTR4-7 promoter. Figure 10. Fluorescence of the seven rice gene HrR7J promoter.
A: pHTR711-l启动子的荧光图；  A: Fluorescence map of the pHTR711-1 promoter;
B : pHTR711-2启动子的荧光图；  B: Fluorescence map of the pHTR711-2 promoter;
C: pHTR711-3启动子的荧光图；  C: Fluorescence diagram of the pHTR711-3 promoter;
D : pHTR711-4启动子的荧光图；  D: Fluorescence of the pHTR711-4 promoter;
E: pHTR711-5启动子的荧光图；  E: Fluorescence of the pHTR711-5 promoter;
F: pHTR711-6启动子的荧光图；  F: Fluorescence diagram of the pHTR711-6 promoter;
G: pHTR711-7启动子的荧光图。 图 11、 7个拟南芥基因 HTR4和 7个水稻基因 HTR711启动子荧光值的箱体图。 A: 7个拟南芥基因 H7 W启动子荧光值的箱体图；  G: Fluorescence of the pHTR711-7 promoter. Figure 11. Box diagram of the fluorescence values of the seven Arabidopsis gene HTR4 and seven rice gene HTR711 promoters. A: a box diagram of the fluorescence values of seven Arabidopsis genes H7 W promoter;
B : 7个拟南芥基因 HrR7 启动子荧光值的箱体图。 图 12、 拟南芥基因腿1、 ΗΊΈ4启动子及 CaMV35S启动子驱动 Gt/S基因表达图。 A: pCambial300-pHTRl-GUS转化拟南芥， 在整株中 GUS组织化学染色结果； B : pCambial300-CaMV35S-GUS转化拟南芥， 在整株中 GUS组织化学染色结果； C: pCambial300-pHTR4-GUS转化拟南芥， 在整株中 GUS组织化学染色结果。 图 13、 玉米基因 HTTWO^？、 HTR102、 HrR 4和 HrR 3启动子驱动 Gt/S基因表达 图。  B: Box diagram of the fluorescence values of seven Arabidopsis gene HrR7 promoters. Figure 12. Arabidopsis thaliana gene leg 1, ΗΊΈ4 promoter and CaMV35S promoter drive Gt/S gene expression map. A: pCambial300-pHTRl-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in whole plants; B: pCambial300-CaMV35S-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in whole plants; C: pCambial300-pHTR4- GUS transforms Arabidopsis thaliana, GUS histochemical staining results in whole plants. Figure 13. Maize gene HTTWO^? The HTR102, HrR 4 and HrR 3 promoters drive the Gt/S gene expression map.
Al， A2: pCambial300-pHTR101-GUS转化拟南芥，在拟南芥茎生叶和莲座叶中 GUS 组织化学染色结果；  Al, A2: pCambial300-pHTR101-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in Arabidopsis stem leaves and rosette leaves;
Bl， B2: PCambial300- pHTR102-GUS转化拟南芥，在拟南芥茎生叶和莲座叶中 GUS 组织化学染色结果； Bl, B2: P Cambial300-pHTR102-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in Arabidopsis stem leaves and rosette leaves;
Cl， C2: PCambial300- pHTR104-GUS转化拟南芥，在拟南芥茎生叶和莲座叶中 GUS 组织化学染色结果； Cl, C2: P Cambial300-pHTR104-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in Arabidopsis stem leaves and rosette leaves;
Dl , D2: PCambial300- pHTR113-GUS转化拟南芥，在拟南芥茎生叶和莲座叶中 GUS 组织化学染色结果。 图 14、 水稻基因 HrR7(½启动子驱动 Gt/S基因表达图。 Dl, D2: P Cambial300-pHTR113-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in Arabidopsis stem leaves and rosette leaves. Figure 14. Rice gene HrR7 (1⁄2 promoter drives Gt/S gene expression map).
A: pCambial300-pHTR706-GUS转化拟南芥， 在茎生叶中 GUS组织化学染色结果； B : PCambial300- pHTR706-GUS转化拟南芥， 在莲座叶中 GUS组织化学染色结果。 具体实施方式 A: pCambial300-pHTR706-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in stem leaves; B: P Cambial300-pHTR706-GUS transformed Arabidopsis thaliana, GUS histochemical staining results in rosette leaves. detailed description
本发明人通过广泛而深入的研究， 揭示一种核酸， 将其作为启动子元件， 可指导目 的基因高水平表达。 本发明的启动子来源于 Histone3.3基因的启动子区域。 本发明的启 动子具有广泛的活性， 能够指导目的基因高水平表达 (强表达)。 在此基础上完成了本发 明。 如本文所用， 所述的 "启动子" 或 "启动子区（域） " 是指一种核酸序列， 其通常存 在于目的基因编码序列的上游 (5'端)， 能够引导核酸序列转录为 mRNA。 一般地， 启动 子或启动子区提供 RNA聚合酶和正确起始转录所必需的其它因子的识别位点。 在本文 中， 所述的启动子或启动子区包括启动子的变体， 其通过***或删除调控区域， 进行随 机或定点突变等来获得。 The present inventors have revealed, through extensive and intensive research, a nucleic acid which is used as a promoter element to direct high-level expression of a gene of interest. The promoter of the present invention is derived from the promoter region of the Hist one 3.3 gene. The promoter of the present invention has a wide range of activities and is capable of directing high level expression (strong expression) of the gene of interest. The present invention has been completed on this basis. As used herein, "promoter" or "promoter region (domain)" refers to a nucleic acid sequence that is normally present upstream (5' end) of the coding sequence of the gene of interest and is capable of directing transcription of the nucleic acid sequence into mRNA. . Typically, the promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription. Herein, the promoter or promoter region includes a variant of a promoter which is obtained by inserting or deleting a regulatory region, performing random or site-directed mutagenesis or the like.
如本文所用， "分离的" 是指物质从其原始环境中分离出来 (如果是天然的物质， 原 始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化 的， 但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开， 则为分离纯 化的。  As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
如本文所用， 所述的 "可操作地连接" 或 "操作性连接" 是指两个或多个核酸区域 或核酸序列的功能性的空间排列。 例如： 启动子区被置于相对于目的基因核酸序列的特 定位置， 使得核酸序列的转录受到该启动子区域的引导， 从而， 启动子区域被 "可操作 地连接" 到该核酸序列上。  As used herein, "operably linked" or "operably linked" refers to a spatial arrangement of the functionality of two or more nucleic acid regions or nucleic acid sequences. For example, the promoter region is placed at a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region such that the promoter region is "operably linked" to the nucleic acid sequence.
如本文所用， 所述的 "高水平表达" 是指： 利用本发明的启动子指导一目的基因 (如 GUS基因）的表达与 CaMV35S启动子指导该目的基因表达相比，利用本发明的启动子所 得的表达量显著更高。 蛋白表达量的检测是本领域技术人员熟知的技术。  As used herein, "high level expression" refers to: using the promoter of the present invention to direct expression of a gene of interest (such as the GUS gene) and using the promoter of the present invention as compared to the expression of the CaMV35S promoter to direct the gene of interest. The amount of expression obtained is significantly higher. Detection of the amount of protein expression is a technique well known to those skilled in the art.
如本文所用， 所述的 " SEQ ID NO: 34中（1-743)〜1405位所示的核苷酸序列" 也即 指所述序列可起始于 SEQ ID NO: 34中第 1位至第 743位中的任一位的碱基，终止于 SEQ ID NO: 1中第 1405位的碱基。  As used herein, the "nucleotide sequence shown by (1-743) to 1405 in SEQ ID NO: 34" means that the sequence can start from position 1 in SEQ ID NO: 34 to The base of any of position 743 ends at the 1405th base of SEQ ID NO: 1.
如本文所用， 所述的 " SEQ ID NO: 35中（1-1076)〜1369位所示的核苷酸序列" 也 即指所述序列可起始于 SEQ ID NO: 35中第 1位至第 1076位中的任一位的碱基， 终止 于 SEQ ID NO: 1中第 1369位的碱基。 启动子及其指导的基因表达  As used herein, the "nucleotide sequence shown by (1-1076) to 1369 in SEQ ID NO: 35" means that the sequence can start from position 1 in SEQ ID NO: 35 to The base of any of position 1076 terminates at the 1369th base of SEQ ID NO: 1. Promoter and its directed gene expression
基于本发明人的新发现，提供一种分离的核酸，所述的核酸具有 SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 13、 SEQ ID NO: 14、 SEQ ID NO: 15、 SEQ ID NO: Based on the novel discovery by the present inventors, an isolated nucleic acid having SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:
16、 SEQ ID NO: 17所示的核苷酸序列或这些序列的片段以及含有这些序列的长度更长 的核苷酸序列， 所述的核酸可作为指导目的基因表达的启动子元件。 此外， 本发明还包括上述核酸的一些具有相同功能的变异体。 包括： 16. A nucleotide sequence as shown in SEQ ID NO: 17 or a fragment of these sequences and a nucleotide sequence having a longer length comprising the sequence, the nucleic acid being useful as a promoter element for directing expression of a gene of interest. Furthermore, the present invention also includes some variants of the above nucleic acids having the same function. include:
序列在严格条件下能够与 SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: The sequence is capable of interacting with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: under stringent conditions:
13、 SEQ ID NO: 14、 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17所示的核苷酸序 列或其片段或延长序列杂交且具有指导目的基因高水平表达功能的核酸； 或 13. SEQ ID NO: 14, SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 The nucleotide sequence shown in SEQ ID NO: 17 or a fragment thereof or an extended sequence which hybridizes and has a nucleic acid which directs a high level of expression of the gene of interest;
序列与 SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 13、 SEQ ID NO: SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO:
14、 SEQ ID NO: 15、 SEQ ID NO: 16、 SEQ ID NO: 17所示的核苷酸序列或其片段或延 长序列有 80%以上同源性且具有指导目的基因高水平表达功能的核酸； 或 14. Nucleic acid having a nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or a fragment or an extended sequence thereof having 80% or more homology and having a high level of expression function indicative of a gene of interest ; or
序列与 SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 13、 SEQ ID NO: 14、 SEQ ID NO: 15、 SEQ ID NO: 16、 SEQ ID NO: 17所示的核苷酸序列或其片段或延 长序列互补 (优选完全互补）的核酸。  SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: A nucleic acid of the indicated nucleotide sequence or a fragment thereof or an extended sequence that is complementary (preferably fully complementary).
多核苷酸的杂交是本领域技术人员熟知的技术， 特定的一对核酸的杂交特性指示它 们的相似性或同一性。因此，本发明还涉及与 SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 13、 SEQ ID NO: 14、 SEQ ID NO: 15、 SEQ ID NO: 16、 SEQ ID NO: 17 所示的核苷酸序列或其片段或延长序列杂交且两个序列之间具有至少 50%， 较佳地至少 70% , 更佳地至少 80% (例如 85%、 90% 95% 96%、 97% , 98% 或 99%) 相同性的 核酸。 本发明特别涉及在严格条件下与本发明所述核酸可杂交的多核苷酸。  Hybridization of polynucleotides is a technique well known to those skilled in the art, and the hybridization characteristics of a particular pair of nucleic acids indicate their similarity or identity. Accordingly, the present invention also relates to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ The nucleotide sequence shown by ID NO: 17 or a fragment or extension thereof hybridizes and has at least 50%, preferably at least 70%, more preferably at least 80% (e.g., 85%, 90% 95) between the two sequences. % 96%, 97%, 98% or 99%) Nucleic acids of identity. The invention particularly relates to polynucleotides that hybridize to the nucleic acids of the invention under stringent conditions.
在本发明中， "严格条件" 是指： （1)在较低离子强度和较高温度下的杂交和洗脱， 如 0.2xSSC， 0.1 SDS , 60 °C ; 或 (2)杂交时加有变性剂， 如 50%(v/v)甲酰胺， 0.1%小牛 血清 /0.1%Ficoll， 42°C等； 或 (3)仅在两条序列之间的相同性至少在 90%以上， 更好是 95% 以上时才发生杂交。 并且， 可杂交的核酸也具有指导目的基因高水平表达的功能。 In the present invention, "stringent conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1 SDS, 60 °C ; or (2) hybridization a denaturant such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) only at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%. Moreover, the hybridizable nucleic acid also has a function of directing high level expression of the gene of interest.
在本发明的实例中， 在本发明的启动子的指导下， 可以使 GUS基因或 GFP基因高水 平地表达。 因此可见， 本发明的启动子是一种特别适合于指导目的基因表达的启动子， 在基因表达的研究中具有重要的应用价值。  In the examples of the present invention, the GUS gene or the GFP gene can be expressed at a high level under the guidance of the promoter of the present invention. Therefore, it can be seen that the promoter of the present invention is a promoter which is particularly suitable for guiding the expression of a gene of interest, and has important application value in the research of gene expression.
本发明的启动子可以被可操作地连接到目的基因上， 该目的基因相对于启动子而言 可以是外源 (异源）的。 所述的目的基因通常可以是任何核酸序列 (如一种结构性核酸序 列)， 所述的目的基因优选编码具有特定功能的蛋白， 例如某些具有重要特性或功能的蛋 白。  The promoter of the present invention can be operably linked to a gene of interest which can be foreign (heterologous) relative to the promoter. The gene of interest may generally be any nucleic acid sequence (e.g., a structural nucleic acid sequence), and the gene of interest preferably encodes a protein having a particular function, such as certain proteins having important properties or functions.
例如， 所述的目的基因包括但不限于： GUS基因、 GFP基因、 荧光素酶基因等。 GUS作为一种代表性的指示基因表达状况的工具，可良好地指示由启动子指导的基因表 达情况。 而由本发明的启动子可指导 GUS高表达这一实例， 可显而易见地得知本发明 的启动子作为基因调控元件， 可指导其它任何合适的基因的高表达。  For example, the gene of interest includes, but is not limited to, a GUS gene, a GFP gene, a luciferase gene, and the like. As a representative tool for indicating gene expression status, GUS is a good indicator of gene expression guided by the promoter. While the promoter of the present invention can direct the expression of GUS high expression, it is apparent that the promoter of the present invention as a gene regulatory element can direct the high expression of any other suitable gene.
作为本发明的优选方式， 所述的目的基因可以是一种对于某一植物或特定植物组织 或器官而言缺失或表达量不足的基因， 可将该目的基因与本发明的启动子可操作地连 接， 或将目的基因与本发明的启动子可操作的连接入合适的载体中， 采取适当的方式导 入到适当的细胞内， 并传递到该植物或特定植物组织或器官内， 从而驱动目的基因高水 平地表达。 As a preferred mode of the present invention, the gene of interest may be a gene lacking or insufficiently expressed in a certain plant or a specific plant tissue or organ, and the gene of interest and the promoter of the present invention may be operably Linking, or operably linking the gene of interest to the promoter of the present invention into a suitable vector, introducing it into an appropriate cell in an appropriate manner, and delivering it to the plant or a specific plant tissue or organ, thereby driving the gene of interest. High water Flat expression.
所述的 "植物" 没有特别的限制， 只要该植物适合进行基因的转化操作（转基因操 作）， 如各种农作物、 花卉植物、 或林业植物等。 所述的植物比如可以是： 双子叶植物、 单子叶植物、 或裸子植物。 例如但不限于： 十字花科（如芸薹属的大白菜、 小白菜）， 十 字花科（如鼠耳芥属植物（如拟南芥））， 禾本科（如水稻）， 茄科（如烟草）， 此外还包括瓜 果、 蔬菜、 油菜等等。 更具体地， 所述的植物包括（但不限于）： 小麦、 大麦、 黑麦、 水 稻、 玉米、 高梁、 甜菜、 苹果、 梨、 李、 桃、 杏、 樱桃、 草莓、 木莓、 黑莓、 豆、 扁豆、 豌豆、 大豆、 油菜、 芥、 罂粟、 齐墩果、 向日葵、 椰子、 蓖麻油植物、 可可豆、 花生、 葫芦、 黄瓜、 西瓜、 棉花、 亚麻、 ***、 黄麻、 柑桔、 柠檬、 葡萄柚、 菠菜、 苘苣、 芦 笋、 洋白菜、 大白菜、 小白菜、 胡萝卜、 洋葱、 土豆、 西红柿、 青椒、 鳄梨、 桂皮、 樟 脑、 烟叶、 坚果、 咖啡、 茄子、 甘蔗、 茶叶、 胡椒、 葡萄树、 蚝麻草、 香蕉、 天然橡胶 树和观赏植物等。  The "plant" is not particularly limited as long as the plant is suitable for a genetic transformation operation (transgenic operation) such as various crops, flower plants, or forestry plants. The plant may be, for example, a dicot, a monocot, or a gymnosperm. For example, but not limited to: cruciferous (such as Chinese cabbage, Chinese cabbage), cruciferous (such as Arabidopsis (such as Arabidopsis), Gramineae (such as rice), Solanaceae (such as Tobacco), in addition to fruits, vegetables, rapeseed, etc. More specifically, the plants include, but are not limited to: wheat, barley, rye, rice, corn, sorghum, beets, apples, pears, plums, peaches, apricots, cherries, strawberries, raspberries, blackberries, beans , lentils, peas, soybeans, rapeseed, mustard, poppy, olean, sunflower, coconut, castor oil plant, cocoa beans, peanuts, gourd, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, grape Pomelo, spinach, chicory, asparagus, cabbage, Chinese cabbage, pakchoi, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugar cane, tea, pepper, vine , ramie, bananas, natural rubber trees and ornamental plants.
本发明的启动子还可以被可操作地连接到被改进的目的基因序列上， 该目的基因相 对于启动子是外源 (异源)的。 所述的目的基因可以被改进来产生各种期望的特性。例如， 目的基因可以被改进来增加必需氨基酸的含量， 提高氨基酸序列的翻译， 改变翻译后的 修饰 (如磷酸化位点）， 将翻译产物转运到细胞外， 改善蛋白的稳定性， ***或删除细胞 信号等。  The promoter of the present invention may also be operably linked to a modified gene sequence of interest which is exogenous (heterologous) relative to the promoter. The gene of interest can be modified to produce a variety of desirable properties. For example, the gene of interest can be modified to increase the amount of essential amino acids, increase translation of amino acid sequences, alter post-translational modifications (such as phosphorylation sites), transport translation products out of the cell, improve protein stability, insert or delete Cell signal, etc.
此外， 启动子和目的基因可以设计成下调特定基因。 这一般是通过将启动子连接到 目的基因序列上来实现， 该序列以反义反向被引导。 本领域的普通技术人员熟悉这种反 义技术。 任何核酸序列可以以这种方式被调节。 本发明的启动子和目的基因序列可被包含在重组载体中。  In addition, promoters and genes of interest can be designed to downregulate specific genes. This is typically accomplished by ligating the promoter to the sequence of the gene of interest, which sequence is directed in antisense orientation. Those of ordinary skill in the art are familiar with such antisense techniques. Any nucleic acid sequence can be modulated in this manner. The promoter and gene of interest sequences of the invention can be included in a recombinant vector.
所述的重组载体一般包括 (从 5'到 3'方向）： 引导目的基因转录的启动子， 和目的基 因。 如果需要， 所述的重组载体还可以包括 3'转录终止子， 3'多聚核苷酸化信号， 其它 非翻译核酸序列， 转运和靶向核酸序列、 抗性选择标记、 增强子或操作子。  The recombinant vector generally includes (from the 5' to 3' direction): a promoter that directs transcription of the gene of interest, and a gene of interest. If desired, the recombinant vector may also include a 3' transcriptional terminator, a 3' polynucleotideization signal, other non-translated nucleic acid sequences, a transport and targeting nucleic acid sequence, a resistance selection marker, an enhancer or an operator.
作为一种方式， 所述的重组载体包括本发明的启动子， 在所述启动子的下游包含多 克隆位点或至少一个酶切位点。 当需要表达目的基因时， 将目的基因连接入适合的多克 隆位点或酶切位点内， 从而将目的基因与启动子可操作地连接。  In one embodiment, the recombinant vector comprises a promoter of the invention comprising a cloning site or at least one cleavage site downstream of the promoter. When expression of the gene of interest is desired, the gene of interest is ligated into a suitable polycloning site or restriction site to operably link the gene of interest to the promoter.
用于制备重组载体的方法是本领域技术人员所熟知的。 术语 "表达载体" 指本领域 熟知的细菌质粒、 噬菌体、 酵母质粒、 病毒或其他载体。 总之， 只要其能够在宿主体内 复制和稳定， 任何质粒和载体都是可以被采用的。 优选的， 所述的表达载体是真核表达 载体。  Methods for preparing recombinant vectors are well known to those skilled in the art. The term "expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, virus or other vector well known in the art. In summary, any plasmid and vector can be employed as long as it is capable of replication and stabilization in the host. Preferably, the expression vector is an eukaryotic expression vector.
本领域的技术人员熟知的方法能用于构建含有本发明所述的启动子和 /或目的基因 序列的表达载体。这些方法包括体外重组 DNA技术、 DNA合成技术、体内重组技术等。 表达载体还包括翻译起始用的核糖体结合位点和转录终止子。 此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转化的宿 主细胞的表型性状， 如二氢叶酸还原酶、 新霉素抗性、 潮霉素抗性以及绿色荧光蛋白Methods well known to those skilled in the art can be used to construct expression vectors containing the promoters and/or gene sequences of interest described herein. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green Fluorescent protein
(GFP) GUS等。 (GFP) GUS et al.
重组载体中除了含有本发明的启动子， 还可含有一种或多种其它启动子。 所述的其 它启动子例如是： 组织特异性的、 组成型的或诱导型的。 例如甘露氨酸合成酶的花椰菜 花叶病毒 19S和 35S(CaMV19S CaMV35S)、 增强的 CaMV、 烟草 RB7等。  The recombinant vector may contain one or more other promoters in addition to the promoter of the present invention. Such other promoters are, for example, tissue-specific, constitutive or inducible. For example, mannose synthase broccoli mosaic virus 19S and 35S (CaMV19S CaMV35S), enhanced CaMV, tobacco RB7 and the like.
作为本发明的一种实例， 所述的载体是 pCambia系列载体。 即利用 pCambia上的多 克隆位点， 可将本发明的启动子区域构建到报告基因如 GUS或 GFP的前面， 转化宿主 细胞， 启动子激活 GUS或 GFP编码基因的表达， 所述启动受到启动子区各顺式作用元 件的调控， 模拟了基因在体内被激活转录的状况。 包含上述适当的启动子和目的基因的载体， 可以用于转化适当的宿主细胞， 以使其 能够表达蛋白质。  As an example of the present invention, the vector is a pCambia series vector. That is, using the multiple cloning site on pCambia, the promoter region of the present invention can be constructed in front of a reporter gene such as GUS or GFP, and the host cell can be transformed, and the promoter activates expression of a GUS or GFP-encoding gene, and the promoter is activated by a promoter. The regulation of each cis-acting element in the region mimics the state in which the gene is activated and transcribed in vivo. A vector comprising the appropriate promoter and gene of interest described above can be used to transform a suitable host cell to enable expression of the protein.
宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高 等真核细胞， 如植物细胞。 代表性例子有： 大肠杆菌， 酵母， 动物的组织细胞， 植物细 胞等。 本领域一般技术人员都清楚如何选择适当的载体和宿主细胞。  The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples are: Escherichia coli, yeast, animal tissue cells, plant cells, and the like. It will be apparent to one of ordinary skill in the art how to select an appropriate vector and host cell.
本发明的多核苷酸在高等真核细胞中表达时， 如果在载体中***增强子序列时将会 使转录得到增强。 增强子是 DNA的顺式作用因子， 通常大约有 10到 300个碱基对， 作 用于启动子以增强基因的转录。  When a polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, used as a promoter to enhance transcription of a gene.
本领域一般技术人员都清楚如何选择适当的载体、 启动子、 增强子或宿主细胞。 用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 当宿主为原 核生物如大肠杆菌时， 能吸收 DNA 的感受态细胞可在指数生长期后收获， 用 CaCl2法 处理， 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl2。 如果需要， 转化也可 用电穿孔的方法进行。 当宿主是真核生物， 可选用如下的 DNA转染方法： 磷酸钙共沉 淀法， 常规机械方法如显微注射、 电穿孔、 脂质体包装等。 转化植物也可使用农杆菌转 化或基因枪转化等方法， 例如叶盘法、 幼胚转化法、 花芽浸泡法等。 对于转化的植物组 织或器官可以用常规方法再生成植株， 从而获得转基因的植物。 It will be apparent to one of ordinary skill in the art how to select an appropriate vector, promoter, enhancer or host cell. Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art. Another method is to use MgCl 2 . Conversion can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like. The transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, immature embryo transformation method, flower bud soaking method and the like. For transformed plant tissues or organs, plants can be regenerated by conventional methods to obtain transgenic plants.
作为一种方式， 制备转基因植物的方法是： 将携带启动子和目的基因（两者可操作 地连接）的双元载体转入农杆菌， 农杆菌再将含启动子和目的基因的载体片段整合到植 物的染色体上。 涉及的转基因受体植物例如是拟南芥、 烟草和水稻。 本发明的主要优点在于：  As a method, a method for preparing a transgenic plant is: transferring a binary vector carrying a promoter and a gene of interest (both operably linked) into Agrobacterium, and Agrobacterium further integrates a vector fragment containing a promoter and a gene of interest Go to the chromosome of the plant. The transgenic receptor plants involved are, for example, Arabidopsis thaliana, tobacco and rice. The main advantages of the invention are:
(1) 揭示一种可指导目的基因高水平表达的启动子， 利用本发明的启动子可以高强 度地启动目的基因的大量表达。  (1) A promoter which can direct high-level expression of a gene of interest is revealed, and a large amount of expression of a gene of interest can be efficiently initiated by the promoter of the present invention.
(2) 本发明为外源基因表达导致相关基因沉默的现象以及水稻育种过程中引进的外 源启动子如 Cauliflower Mosaic Virus 35S Promoter (CaMV 35S)存在潜在的生物安全性 风险等问题提供了一种新的解决途径。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件如 J.萨姆布鲁克等编著， 分子克隆实验指南， 第三版， 科学出版社， 2002中所述的 条件， 或按照制造厂商所建议的条件。 除非另外说明， 否则百分比和份数按重量计算。 (2) The present invention is a phenomenon in which the expression of a foreign gene leads to the silencing of a related gene and the introduction of rice during the breeding process. Source promoters such as Cauliflower Mosaic Virus 35S Promoter (CaMV 35S) provide a new solution to the potential biosafety risks. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experimental Guide, Third Edition, Science Press, 2002, or according to the manufacturer. The suggested conditions. Percentages and parts are by weight unless otherwise stated.
除非另行定义， 文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义 相同。 此外， 任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。 文中所 述的较佳实施方法与材料仅作示范之用。 实施例 1、 启动子的获得  Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only. Example 1. Acquisition of promoter
本发明分别克隆了拟南芥组蛋白变体 H3.3基因 HrR4(AT4G40030)以及水稻组蛋白 基因 HrR7 (LOC_Os03g27310)和 HTT^ ^OC—OsC^gCMC O)的 5'端调控区启动子。 拟 南芥基因 H7 W启动子序列如 SEQ ID NO: 2;水稻基因 HrR7 启动子序列如 SEQ ID NO: 3；水稻基因 HTR712启动子序列如 SEQ ID NO: 4；拟南芥基因 HTR1启动子序列如 SEQ ID NO: 1， 作为对照。  The 5'-end regulatory region promoter of the Arabidopsis thaliana histone variant H3.3 gene HrR4 (AT4G40030) and the rice histone genes HrR7 (LOC_Os03g27310) and HTT^^OC-OsC^gCMC O) were cloned. The Arabidopsis gene H7 W promoter sequence is SEQ ID NO: 2; the rice gene HrR7 promoter sequence is SEQ ID NO: 3; the rice gene HTR712 promoter sequence is SEQ ID NO: 4; the Arabidopsis gene HTR1 promoter sequence As SEQ ID NO: 1, as a control.
上述启动子序列可用于构建报告基因表达载体。 实施例 2、 拟南芥基因 HrR4启动子驱动 GUS基因表达的活性检测  The above promoter sequences can be used to construct reporter gene expression vectors. Example 2. Arabidopsis thaliana gene HrR4 promoter drives the activity assay of GUS gene expression
1、 转基因烟草 GUS组织化学染色检测启动子活性  1. Transgenic tobacco GUS histochemical staining detection promoter activity
在拟南芥基因 HTI 和 HTR4启动子、 CaMV35S的驱动下， 以 GUS为报告基因构 建植物表达载体 pHTRl-GUS、 pHTR4-GUS及 CaMV35S-GUS。  The plant expression vectors pHTRl-GUS, pHTR4-GUS and CaMV35S-GUS were constructed using GUS as a reporter gene driven by the Arabidopsis thaliana HTI and HTR4 promoters and CaMV35S.
拟南芥基因 HTR1、 启动子、 CaMV35S序列分别***到 pCambial300表达载 体 (购自 Cambia公司）的 Hindlll和 BamHI酶切位点中（即位于 GUS报告基因的上游）。 GUS元件***到 pCambial300的 B mH/禾卩 Sac/酶切位点。  The Arabidopsis thaliana gene HTR1, promoter, and CaMV35S sequences were inserted into the Hindlll and BamHI restriction sites of the pCambial300 expression vector (purchased from Cambia) (i.e., upstream of the GUS reporter gene). The GUS element was inserted into the B mH/he Sac/cleavage site of pCambial300.
获得的重组载体分别命名为： pCambial 300-pHTRl-GUS、 pCambial 300-pHTR4-GUS 及 pCambial300-CaMV35S-GUS。  The recombinant vectors obtained were designated as: pCambial 300-pHTRl-GUS, pCambial 300-pHTR4-GUS and pCambial300-CaMV35S-GUS.
将构建的重组载体转化农杆菌 EH4105(参见 Hood, E. E.等， Transgenic Res., 1993, 2, 208 - 218) , 筛选阳性克隆。 以获得的含有目的基因质粒的农杆菌转染烟草叶片， 共培养 2天后转入筛选培养基， 去除残余的农杆菌。 大约 1个月左右将感染了农杆菌的阳性愈 伤组织进行组织化学染色， 检测 GUS 基因的瞬时表达。 待烟草长出抗性不定芽之后， 将芽切下转入含有抗性的生根培养基中， 约一个月左右将生根的烟草小苗转入盆中， 在 温室中培养， 1个月左右取叶片进行组织化学染色检测 Gt/S基因的瞬时表达。  The constructed recombinant vector was transformed into Agrobacterium EH4105 (see Hood, E. E. et al., Transgenic Res., 1993, 2, 208-218), and positive clones were screened. The obtained Agrobacterium containing the plasmid of the target gene was transfected into tobacco leaves, and after cocultivation for 2 days, it was transferred to a screening medium to remove residual Agrobacterium. The positive callus infected with Agrobacterium was histochemically stained for about one month to detect the transient expression of the GUS gene. After the tobacco grows resistant to adventitious buds, the buds are cut into the rooting medium containing the resistance, and the rooted tobacco seedlings are transferred to the pots in about one month, and cultured in the greenhouse, and the leaves are taken in about 1 month. Histochemical staining was performed to detect the transient expression of the Gt/S gene.
将共培养的愈伤组织或叶片用灭菌蒸馏水漂洗后置于 1.5ml离心管中， 加入没过愈 伤组织的 GUS染色液并混匀， 使愈伤组织与染色液充分接触； 于 37 °C温浴 2小时或过 夜； 以 70%的乙醇为脱色液对愈伤组织进行脱色并观察蓝色斑点。 The co-cultured callus or leaves are rinsed with sterile distilled water and placed in a 1.5 ml centrifuge tube. The GUS staining solution of the wound tissue was mixed and the callus was fully contacted with the staining solution; the bath was incubated at 37 ° C for 2 hours or overnight; the callus was decolorized with 70% ethanol as a decolorizing solution and blue spots were observed.
HrR4(H3.3)的启动子主要在细胞间期高度表达， 在营养器官高水平组成性表达。 HTR1(H3.1)的表达是与 DNA复制紧密相关的， 在 DNA复制过程中即 S期 H3.1大量合 成。 GUS 组织化学染色结果表明了在烟草愈伤组织中拟南芥基因 HTR1 启动子驱动的 Gt/S基因的表达活性明显高于 HTR4和 CaMV35S驱动的 Gt/S表达活性， 如图 1所示。  The promoter of HrR4 (H3.3) is highly expressed in the intercellular phase and is constitutively expressed at high levels in vegetative organs. The expression of HTR1 (H3.1) is closely related to DNA replication, and a large number of S phase H3.1 is synthesized during DNA replication. The results of GUS histochemical staining indicated that the expression of Gt/S gene driven by HTR1 promoter in Arabidopsis thaliana was significantly higher than that of HTR4 and CaMV35S-driven Gt/S expression, as shown in Figure 1.
在转基因烟草叶片中，拟南芥基因 H7 W启动子驱动的 Gt/S基因的表达活性明显高 于 HTR1驱动的 表达活性， 如图 2所示。 但是， 在不同时期， 拟南芥基因 HTR1和 H7 W启动子的驱动活性均比常用的组成型启动子 CaMV35S的活性高。  In the leaves of transgenic tobacco, the expression activity of the Gt/S gene driven by the H7 W promoter of Arabidopsis thaliana gene was significantly higher than that of HTR1, as shown in Figure 2. However, at different stages, the Arabidopsis gene HTR1 and H7 W promoters were more active than the commonly used constitutive promoter CaMV35S.
2、 转基因拟南芥 GUS组织化学染色检测启动子活性 2, transgenic Arabidopsis GUS histochemical staining detection promoter activity
将构建的植物表达载体 pCambial300-pHTRl-GUS、 PCambial300-pHTR4- GUS 及 PCambial300-CaMV35S-GUS转化农杆菌 GV W(Invitrogen)， 筛选阳性克隆。 以获得的 含有目的基因质粒的农杆菌转染拟南芥花序， 用保鲜膜包裹拟南芥苗后暗培养 2天， 之 后转入光培养， 在温室培养约 1个半月后收集种子。 在筛选培养基上筛选 TO代种子， 鉴定 T1代苗后转入盆中， 在温室培养 2周后， 取叶片进行组织化学染色检测 Gt/S基因 的瞬时表达。 The constructed plant expression vectors pCambial300-pHTRl-GUS, P Cambial300-pHTR4-GUS and P Cambial300-CaMV35S-GUS were transformed into Agrobacterium GV W (Invitrogen), and positive clones were selected. The obtained Agrobacterium containing the plasmid of the target gene was transfected into the Arabidopsis inflorescence, and the Arabidopsis thaliana seedlings were wrapped in plastic wrap for 2 days, then transferred to light culture, and the seeds were collected after about one and a half months in the greenhouse culture. The TO seeds were screened on the screening medium, and the T1 generation seedlings were identified and transferred into pots. After 2 weeks of greenhouse culture, the leaves were subjected to histochemical staining to detect the transient expression of Gt/S gene.
在转基因拟南芥叶片 (尤其在花序)中， 拟南芥基因 H7 W启动子驱动的 Gt/S基因的 表达活性明显高于 HTR1和 CaMV35S驱动的 GUS表达活性， GUS染色最明显， 如图 3 所示。 以上实验说明在不同生长时期， 拟南芥基因 H7 W启动子的驱动活性较常用的组 成型启动子 CaMV35S的活性高。  In the transgenic Arabidopsis leaves (especially in inflorescence), the expression of the Gt/S gene driven by the Arabidopsis thaliana gene H7 W promoter was significantly higher than that of HTR1 and CaMV35S-driven GUS expression, and the GUS staining was most obvious, as shown in Fig. 3. Shown. The above experiments indicated that the driving activity of the H7 W promoter of Arabidopsis thaliana gene was higher than that of the commonly used assembly promoter CaMV35S at different growth stages.
3、 定量 RT-PCR(qRT-PCR)方法检测启动子活性 3. Quantitative RT-PCR (qRT-PCR) method for detecting promoter activity
构建植物表达载体 pCambial301-CaMV35S-Copl-YFP: 将 CaMV35S启动子片段克 隆到载体 pCambial301(购自 Cambia公司）的 ffi/^/H和 B mH/酶切位点；之后将 copl (以 拟 南 芥 cDNA 为 模 板 ， 以 sall-copl-5 ： Construction of plant expression vector pCambial301-CaMV35S-Copl-YFP: The CaMV35S promoter fragment was cloned into the ffi/^/H and B mH/ restriction sites of vector pCambial 301 (purchased from Cambia); copl (with Arabidopsis thaliana) cDNA is a template to sall-copl-5:
CCCGTCGACATGGAAGAGATTTCGACGGATCCGG (SEQ ID NO: 5)； speI-copl-3 : CCCACTAGTCGCAGCGAGTACCAGAACTTTG (SEQ ID NO: 6)为引物的 PCR扩增产物) 片段克隆到载体上的 禾 B Spe/位点； 将 YFP片段克隆到载体的 Spe/和 S^/位点。 CCCGTCGACATGGAAGAGATTTCGACGGATCCGG (SEQ ID NO: 5); speI-copl-3: CCCACTAGTCGCAGCGAGTACCAGAACTTTG (SEQ ID NO: 6) is the PCR amplification product of the primer) The fragment was cloned into the vector B Spe/site; the YFP fragment was cloned into the vector Spe/ and S^/sites.
将构建的植物表达载体 pCambial301-CaMV35S-Copl-YFP 转化农杆菌 Transformation of a plant expression vector pCambial301-CaMV35S-Copl-YFP into Agrobacterium
GyJiOi(Invitrogen) , 筛选阳性克隆。 以获得的含有目的基因质粒的农杆菌转染拟南芥花 序， 用保鲜膜包裹拟南芥苗后暗培养 2天， 之后转入光培养， 在温室培养约 1个半月后 收集种子。 在筛选培养基上筛选 T0代种子， 鉴定 T1代苗后转入盆中， 在温室培养 2周 后， 取叶片抽提 RNA， 反转录成 cDNA后做定量 PCR反应。 GyJiOi (Invitrogen), screening positive clones. The obtained Agrobacterium containing the plasmid of the target gene was transfected into the Arabidopsis thaliana, and the Arabidopsis thaliana seedlings were wrapped in plastic wrap for 2 days, then transferred to light culture, and the seeds were collected after about one and a half months in the greenhouse. The T0 generation seeds were screened on the screening medium, and the T1 generation seedlings were identified and transferred to the pots. After 2 weeks of culture in the greenhouse, the leaves were extracted for RNA and reverse transcribed into cDNA for quantitative PCR reaction.
HTR1和 H7 W启动子（内源）的表达水平可以通过检测 HTR1和 HTR4基因 cDNA的 表达来反映，因为 Copl是拟南芥的内源基因，所以通过检测 YFP的活性来反映 CaMV35S 启动子的表达水平。 The expression levels of the HTR1 and H7 W promoters (endogenous) can be detected by detecting the cDNA of HTR1 and HTR4 genes. Expression reflects that since Copl is an endogenous gene of Arabidopsis thaliana, the expression level of the CaMV35S promoter is reflected by detecting the activity of YFP.
RT-PCR检测引物如下：  RT-PCR detection primers are as follows:
HTR1： F: CGTACCAAGCAAACCGCAAGGAAA(SEQ ID NO: 7)；  HTR1: F: CGTACCAAGCAAACCGCAAGGAAA (SEQ ID NO: 7);
R: AGGACGGAATCTGTGTGGCTTCTT(SEQ ID NO: 8)；  R: AGGACGGAATCTGTGTGGCTTCTT (SEQ ID NO: 8);
HTR4-. F: TGCACCAACTACTGGTGGAGTCAA(SEQ ID NO: 9)；  HTR4-. F: TGCACCAACTACTGGTGGAGTCAA (SEQ ID NO: 9);
R: AGCTAAGACAGCATGGCTCTGGAA(SEQ ID NO: 10)；  R: AGCTAAGACAGCATGGCTCTGGAA (SEQ ID NO: 10);
YFP: F: TGACCCTGAAGTTCATCTGCACCA(SEQ ID NO: 11)；  YFP: F: TGACCCTGAAGTTCATCTGCACCA (SEQ ID NO: 11);
R: TCTTGTAGTTGCCGTCGTCCTTGA(SEQ ID NO: 12)。  R: TCTTGTAGTTGCCGTCGTCCTTGA (SEQ ID NO: 12).
图 4 显示了 HTRl , HTR4， YFP 基因 Ct 值的差异图， 本发明人选取了 3 株转 pCambial301-CaMV35S-Copl-YFP 的转基因拟南芥苗作为材料， 横坐标为 3 个样本 (35S-1 , 35S-2, 35S-3) , 纵坐标为 Ct值。  Figure 4 shows the difference maps of the Ct values of the HTR1, HTR4, and YFP genes. The inventors selected three transgenic Arabidopsis thaliana seedlings transfected with pCambial301-CaMV35S-Copl-YFP as the material, and the abscissa was 3 samples (35S-1). , 35S-2, 35S-3), the ordinate is the Ct value.
定量的结果显示三个材料中， H7 W基因的 Ct值最小， HTR1比 1 ^略低。 这就反 映了在三株转基因拟南芥叶片中， H7 W启动子的表达是最强的。 实施例 3、 水稻基因 HTR 711和 HTR 712启动子驱动 GUS基因表达  Quantitative results showed that among the three materials, the Ht W gene had the smallest Ct value and HTR1 was slightly lower than 1 ^. This reflects the strongest expression of the H7 W promoter in the leaves of three transgenic Arabidopsis thaliana. Example 3. Rice gene HTR 711 and HTR 712 promoter drive GUS gene expression
在水稻基因 HTR711和 HTR712启动子的驱动下， 以 GUS为报告基因构建植物表达 载 体 pCambial300-pHTR711-GUS 、 pCambial300-pHTR712-GUS 及 PCambial300-CaMV35S-GUS , 将构建的质粒转化农杆菌 EH4105 , 筛选阳性克隆。 The plant expression vectors pCambial300-pHTR711-GUS, pCambial300-pHTR712-GUS and P Cambial300-CaMV35S-GUS were constructed with GUS as a reporter gene driven by the rice gene HTR711 and HTR712 promoters. The constructed plasmid was transformed into Agrobacterium EH4105 and screened. Positive clone.
水稻基因 HTR711和 HTR712启动子、 CaMV35S序列分别***到 pCambial300表达 载体的 Η / ///和 BamHI酶切位点中（即位于 GUS报告基因的上游）。 GUS元件***到 pCambial300的 BamH/禾卩 SotJ酶切位点。  The rice gene HTR711 and HTR712 promoters, CaMV35S sequences were inserted into the Η / /// and BamHI restriction sites of the pCambial300 expression vector (ie, upstream of the GUS reporter gene). The GUS element was inserted into the BamH/Whit SotJ restriction site of pCambial300.
以获得的含有目的质粒的农杆菌转染水稻转化受体 (中花 11)的愈伤组织， 共培养两 天后转入筛选培养基， 去除残余的农杆菌； 三天后将感染了农杆菌的阳性愈伤组织进行 组织化学染色检测 GUS 基因的瞬时表达。 将共培养的愈伤组织用灭菌蒸馏水漂洗后置 于 1.5ml离心管中， 加入没过愈伤组织的 GUS染色液并混匀， 使愈伤组织与染色液充分 接触； 于 37 °C温浴 2小时或过夜； 以 70%的乙醇为脱色液对愈伤组织进行脱色并观察蓝 色斑点。  The obtained Agrobacterium containing the plasmid of interest was transfected into the calli of rice transforming receptor (Zhonghua 11), and after co-cultivation for two days, it was transferred to a screening medium to remove residual Agrobacterium; after three days, it was infected with Agrobacterium positive. The callus was subjected to histochemical staining to detect the transient expression of the GUS gene. The co-cultured callus was rinsed with sterile distilled water, placed in a 1.5 ml centrifuge tube, and the GUS staining solution without callus was added and mixed to make the callus fully contact with the staining solution; warm bath at 37 °C 2 hours or overnight; callus was decolorized with 70% ethanol as a decolorizing solution and blue spots were observed.
GUS组织化学染色结果表明，水稻基因 HTR711和 HTR712启动子驱动的 Gt/S基因 的表达活性较 CaMV35S驱动的 GUS表达活性高， 如图 5所示。  The results of GUS histochemical staining showed that the expression of the Gt/S gene driven by the rice gene HTR711 and HTR712 promoter was higher than that of CaMV35S-driven GUS, as shown in Fig. 5.
以上实验说明， 水稻基因 HTR711和 HTR712启动子的驱动活性较常用的组成型启 动子 CaMV35S的活性高。 实施例 4、 水稻基因 HTR 711和 HTR 712启动子驱动 GFP基因瞬间表达分析 在水稻基因 HTR711和 HTR712启动子驱动下， 以绿色荧光蛋白 (GFP)为报告基因， 构建植物表达载体 pCambial300-pHTR711-GFP和 pCambial300-pHTR712-GFP。 The above experiments indicated that the driving activities of the rice gene HTR711 and HTR712 promoters were higher than those of the commonly used constitutive promoter CaMV35S. Example 4: Rice gene HTR 711 and HTR 712 promoter drive GFP gene transient expression analysis Driven by rice gene HTR711 and HTR712 promoter, green fluorescent protein (GFP) is used as a reporter gene. Plant expression vectors pCambial300-pHTR711-GFP and pCambial300-pHTR712-GFP were constructed.
水稻基因 HTR711和 HTR712启动子、 CaMV35S序列分别***到 pCambial300表达 载体的 Hindlll 和 BamHI 酶切位点中（即位于 GFP 基因的上游）。 GFP 元件***到 pCambial300的 BamH/禾卩 SotJ酶切位点。  The rice gene HTR711 and HTR712 promoters and the CaMV35S sequence were inserted into the Hindlll and BamHI restriction sites of the pCambial300 expression vector (i.e., upstream of the GFP gene). The GFP element was inserted into the BamH/Whit SotJ restriction site of pCambial300.
将构建好的质粒转化农杆菌 EH4105 , 筛选阳性克隆。 选择三叶期的狐尾草 (Green foxtail ， Setaria viridis(L.) P. Beauv) 为 瞬 间 表 达 受 体 材 料 。 挑 取 pCambial300-pHTR711-GFP和 pCambial300-pHTR712-GFP阳性菌株 EH4105的单克隆 在 28 °C， 200rpm培养至 OD6TO为 0.8左右。 取 1.5ml菌液于 1.5ml灭菌离心管中， 4°C， 2000rpm 离心 5分钟， 弃上清； 向收集的菌体中加入 lml灭菌蒸馏水， 重悬菌液， 4°C， 2000rpm离心 5分钟， 弃上清。 向收集的菌体中加入灭菌蒸馏水至 OD6TO为 0.8左右用 于注射植物叶片。 取较健康幼嫩的三叶期狐尾草叶片进行注射实验， 将注射叶片区域作 好标记。 两天后取注射区域的叶片进行显微观察实验。 以去卷积荧光显微镜观察 pCambial300-pHTR711-GFP和 pCambial300-pHTR712-GFP质粒在狐尾草叶片中的表达 状况。 取注射区域 l〜2cm2大小的叶片置于加有一滴蒸馏水的载玻片上， 以放大倍数为 60倍的水镜镜头观察 GFP的表达情况。 The constructed plasmid was transformed into Agrobacterium EH4105 and positive clones were screened. The three-leaf stage foxtail (Green foxtail, Setaria viridis (L.) P. Beauv) was selected for transient expression of the receptor material. Monoclones picking pCambial300-pHTR711-GFP and pCambial300-pHTR712-GFP positive strain EH4105 were cultured at 28 ° C, 200 rpm until the OD 6TO was about 0.8. Take 1.5ml of the bacterial solution in a 1.5ml sterile centrifuge tube, centrifuge at 5°C, 2000rpm for 5 minutes, discard the supernatant; add 1ml of sterile distilled water to the collected cells, resuspend the bacterial solution, centrifuge at 4°C, 2000rpm 5 minutes, discard the supernatant. Sterilized distilled water was added to the collected cells to an OD 6TO of about 0.8 for injection of plant leaves. The healthy and young three-leaf stage foxtail leaves were injected for injection, and the injected leaf area was marked. Two days later, the leaves of the injection area were taken for microscopic observation. The expression of pCambial300-pHTR711-GFP and pCambial300-pHTR712-GFP plasmid in foxtail leaves was observed by deconvolutance fluorescence microscopy. The leaves of the size of 1~ 2 cm 2 in the injection area were placed on a glass slide with a drop of distilled water, and the expression of GFP was observed with a water microscope lens with a magnification of 60 times.
如图 6， 在水稻基因 HTR711和 HTR712启动子的驱动下， GFP基因在狐尾草叶片 细胞核内有强的表达。 实施例 5、 拟南芥基因 HTR4以及水稻基因 HTR7U启动子关键位点分析  As shown in Figure 6, driven by the rice gene HTR711 and HTR712 promoter, the GFP gene is strongly expressed in the nucleus of the leaves of the foxtail. Example 5. Analysis of key positions of Arabidopsis thaliana HTR4 and rice gene HTR7U promoter
对于 pHTR4和 pHTR711两个启动子， 分别取长短不一的 7个片段， 以鉴定其关键 位点。 pHTR4和 pHTR711两个启动子各自构建了 7个片段， 见图 7和 8。 分别以拟南 芥和水稻基因组为模板， 以表 1引物 (注： 应用正向引物为 PHTR4-(1〜7)-Kpnl时， 反向 引物均是反向 PHTR4-R-BamHI; 应用正向引物为 PHTR711-(l〜7)-HindIII时， 反向引 物均是反向 PHTR711-R-BamHI)来扩增所述片段，以黄色荧光蛋白 (YFP)为报告基因的植 物表达载体， 各自构建 7个载体， 引物序列见表 1。 For the two promoters, pHTR4 and pHTR711, 7 fragments of different lengths were taken to identify their key sites. Each of the two promoters, pHTR4 and pHTR711, constructed seven fragments, see Figures 7 and 8. The Arabidopsis thaliana and rice genomes were used as templates, and the primers in Table 1 were used. (Note: When the forward primer was P HTR4-(1~7)-Kpnl, the reverse primers were reverse PHTR4-R-BamHI; When the primer is PHTR711-(l~7)-HindIII, the reverse primers are all reverse PHTR711-R-BamHI) to amplify the fragment, and the yellow fluorescent protein (YFP) is used as a plant expression vector for the reporter gene. Seven vectors were constructed, and the primer sequences are shown in Table 1.
表 1、 7个拟南芥基因 HTR4和 7个水稻基因 HTR711启动子引物序列  Table 1. Seven Arabidopsis genes HTR4 and seven rice genes HTR711 promoter primer sequences
Figure imgf000014_0001
正向 PHTR711-l-HindIII gggAAGCTTatgttttcaatggaactcac 26 正向 PHTR711-2-HindIII ggtAAGCTTtgaaaccatcaatttgaatc 27 正向 PHTR711-3-HindIII cccAAGCTTtgtggatgtacagaaatcaa 28 正向 PHTR711-4-HindIII cccAAGCTTTAGTTTGTACCTCCCCTGGG 29 正向 PHTR711-5-HindIII cccAAGCTTTTAGGATTCCCCTTGGGAT 30 正向 PHTR711-6-HindIII cccAAGCTTCCAGGGCTGTGTAGTCCTG 31 正向 PHTR711-7-HindIII cccAAGCTTGGCGGGGTATTGAGATTCTC 32 反向 PHTR711-R-BamHI ggtGGATCCAGCCCTCTCACCACGAATC 33 拟南芥基因 HrR4启动子分别***到 pCambial300表达载体 和 BamHI酶切位 点中（即位于 YFP基因的上游）， YFP元件***到 pCambial300的 和 SotJ酶切位点。 水稻基因 HTR711启动子分别***到 pCambial300表达载体 Hindlll和 BamHI酶切位点 中（即位于 YFP基因的上游）， YFP元件***到 pCambial300的 和 SotJ酶切位点。
Figure imgf000014_0001
Forward PHTR711-l-HindIII gggAAGCTTatgttttcaatggaactcac 26 Forward PHTR711-2-HindIII ggtAAGCTTtgaaaccatcaatttgaatc 27 Forward PHTR711-3-HindIII cccAAGCTTtgtggatgtacagaaatcaa 28 Forward PHTR711-4-HindIII cccAAGCTTTAGTTTGTACCTCCCCTGGG 29 Forward PHTR711-5-HindIII cccAAGCTTTTAGGATTCCCCTTGGGAT 30 Forward PHTR711-6 -HindIII cccAAGCTTCCAGGGCTGTGTAGTCCTG 31 Forward PHTR711-7-HindIII cccAAGCTTGGCGGGGTATTGAGATTCTC 32 Reverse PHTR711-R-BamHI ggtGGATCCAGCCCTCTCACCACGAATC 33 The Arabidopsis thaliana gene HrR4 promoter is inserted into the pCambial300 expression vector and BamHI restriction site (ie upstream of the YFP gene) The YFP element was inserted into the pCambial300 and the SotJ restriction site. The rice gene HTR711 promoter was inserted into the pCambial300 expression vector Hindlll and BamHI restriction sites (i.e., upstream of the YFP gene), and the YFP element was inserted into the pCambial300 and the SotJ restriction site.
将构建好的质粒分别转化农杆菌 GV ， 筛选阳性克隆。 选择烟草 SAW为瞬间表 达受体材料。挑取阳性菌株 GV3101的单克隆在 28°C， 200rpm培养至 OD6TO为 0.8左右。 取 1.5ml菌液于 1.5ml灭菌离心管中， 4°C， 2000rpm离心 5分钟， 弃上清； 向收集的菌 体中加入 lml灭菌蒸馏水， 重悬菌液， 4°C， 2000rpm离心 5分钟， 弃上清。 向收集的 菌体中加入灭菌蒸馏水至 OD6QQ为 0.8左右用于注射植物叶片。 取较健康幼嫩的烟草叶 片进行注射实验， 将注射叶片区域作好标记。 两天后取注射区域的叶片进行显微观察实 验。 以去卷积荧光显微镜观察质粒在烟草叶片中的表达状况。 取注射区域 l〜2cm2大小 的叶片置于加有一滴蒸馏水的载玻片上， 以放大倍数为 60倍的水镜镜头观察 YFP的表 达情况。 7个拟南芥基因 HTR4和 7个水稻基因 HTR711启动子片段的荧光图如图 9和 10所示。计算 7个拟南芥基因 HTR4和 7个水稻基因 HTR711启动子片段 lOxlOpixel的 荧光值绘制箱体图， 如图 11所示。 The constructed plasmids were separately transformed into Agrobacterium GV, and positive clones were screened. Tobacco SAW was selected as a transient expression receptor material. The monoclonal clone carrying the positive strain GV3101 was cultured at 28 ° C, 200 rpm until the OD 6TO was about 0.8. Take 1.5ml of the bacterial solution in a 1.5ml sterile centrifuge tube, centrifuge at 5 °C, 2000rpm for 5 minutes, discard the supernatant; add 1ml of sterile distilled water to the collected cells, resuspend the bacterial solution, centrifuge at 4 ° C, 2000 rpm 5 minutes, discard the supernatant. Sterilized distilled water was added to the collected cells to an OD 6QQ of about 0.8 for injection of plant leaves. Injecting experiments were carried out on healthy and young tobacco leaves, and the area of the injected leaves was marked. Two days later, the leaves of the injection area were taken for microscopic observation. The expression of the plasmid in tobacco leaves was observed by deconvolutance fluorescence microscopy. The leaves of the injection area of l~2cm 2 were placed on a glass slide with a drop of distilled water, and the expression of YFP was observed by a water microscope lens with a magnification of 60 times. Fluorescence patterns of seven Arabidopsis gene HTR4 and seven rice gene HTR711 promoter fragments are shown in Figures 9 and 10. The box values of the fluorescence values of the 7 Arabidopsis gene HTR4 and the 7 rice gene HTR711 promoter fragment lOxlOpixel were calculated, as shown in FIG.
通过图 9-10的荧光值和图 11的箱体图可以得出两个基因 HTR4和 HTR711启动子 的关键区段。 从实验结果可以看到拟南芥基因 H7 W启动子的关键区域位于距离翻译起 始位点 453-663bp， 水稻基因 HTR711 启动子的关键区域位于距离翻译起始位点 201-321bp。 因为拟南芥基因 HTR4 启动子在翻译起始位点和转录起始位点之间有一段 463bp 的 intron, 所以这两个启动子的关键区域都是在距离转录起始位点不到 100bp 的 位置。 实施例 6、 转基因拟南芥 GUS组织化学染色检测启动子活性  The key segments of the two genes HTR4 and HTR711 promoters can be derived from the fluorescence values in Figure 9-10 and the box plot in Figure 11. From the experimental results, it can be seen that the key region of the Arabidopsis thaliana H7 W promoter is located at 453-663 bp from the translation initiation site, and the key region of the rice gene HTR711 promoter is located 201-321 bp from the translation initiation site. Because the Arabidopsis gene HTR4 promoter has a 463 bp intron between the translation initiation site and the transcription initiation site, the key regions of both promoters are less than 100 bp from the transcription start site. position. Example 6. Transgenic Arabidopsis thaliana GUS histochemical staining assay for promoter activity
将前述构建的植物表达载体 pCambial300-pHTRl-GUS、 PCambial300-pHTR4- GUS 及 pCambial300-CaMV35S-GUS转化农杆菌 GV W(Invitrogen)， 筛选阳性克隆。 以获 得的含有目的基因质粒的农杆菌转染拟南芥花序，用保鲜膜包裹拟南芥苗后暗培养 2天， 之后转入光培养， 在温室培养约 1个半月后收集种子。 在筛选培养基上筛选 TO代种子， 鉴定 T1 代苗后转入盆中， 在温室培养收种后将收集的种子铺于筛选培养基中， 培养约 10天后， 分别取 30颗整株进行组织化学染色检测 Gt/S基因的瞬时表达。 The plant expression vectors pCambial300-pHTRl-GUS, P Cambial300-pHTR4-GUS and pCambial300-CaMV35S-GUS constructed as described above were transformed into Agrobacterium GV W (Invitrogen), and positive clones were selected. Obtain The Agrobacterium containing the plasmid of the target gene was transfected into the Arabidopsis inflorescence, and the Arabidopsis thaliana seedlings were wrapped in plastic wrap for 2 days, then transferred to light culture, and the seeds were collected after about one and a half months in the greenhouse. The TO seeds were screened on the screening medium, and the T1 generation seedlings were identified and transferred to the pots. After the greenhouse culture was collected, the collected seeds were placed in the screening medium. After about 10 days, 30 whole plants were taken for tissue culture. Chemical staining was used to detect the transient expression of the Gt/S gene.
在转基因拟南芥整株中， 拟南芥基因 H7 W启动子驱动的 Gt/S基因的表达活性明显 高于 HTR1和 CaMV35S驱动的 GUS表达活性， GUS染色最明显， 如图 12所示。 实施例 7、 转基因拟南芥 GUS组织化学染色检测玉米启动子活性  In the whole transgenic Arabidopsis thaliana, the expression of the Gt/S gene driven by the H7 W promoter of Arabidopsis thaliana gene was significantly higher than that of HTR1 and CaMV35S, and the GUS staining was the most obvious, as shown in Fig. 12. Example 7. Transgenic Arabidopsis thaliana GUS histochemical staining for detection of maize promoter activity
建立以玉米 Η3.3 基因 HTR10 SEQ ID NO: 13)、 HTR102(SEQ ID NO: 14)、 HrRJ 04(SEQ ID NO: 15)、 HrR 3(SEQ ID NO: 16)为启动子， 以 Gt/S为报告基因构建植 物表达载体 pHTR101-GUS、 pHTR102-GUS、 pHTR104-GUS及 pHTR113-GUS。  Established with maize Η3.3 gene HTR10 SEQ ID NO: 13), HTR102 (SEQ ID NO: 14), HrRJ 04 (SEQ ID NO: 15), HrR 3 (SEQ ID NO: 16) as promoter, Gt/ S is a reporter gene constructing plant expression vectors pHTR101-GUS, pHTR102-GUS, pHTR104-GUS and pHTR113-GUS.
玉米基 S ΗΊΈ〗0〗、ΗΊΈ〗02、HTW04和 HTR ^？启动子序列分别***到 pCambial300 表达载体 (购自 Cambia公司）的 Η / ///和 BamH/酶切位点中（即位于 GUS报告基因的上 游）。 GUS元件***到 pCambial300的 B mH/禾卩 SotJ酶切位点。  Corn based S ΗΊΈ〗 0, ΗΊΈ 02 02, HTW04 and HTR ^? The promoter sequences were inserted into the Η / / / / and BamH / restriction sites of the pCambial300 expression vector (purchased from Cambia) (i.e., upstream of the GUS reporter gene). The GUS element was inserted into the B mH/ and SotJ cleavage site of pCambial300.
获 得 的 重 组 载 体 分 别 命 名 为 ： pCambial300-pHTR101-GUS 、 pCambial 300-pHTR102-GUS 、 pCambial300-pHTR104-GUS 及 pCambial300- pHTR113-GUS。  The obtained recombinant vectors were named as: pCambial300-pHTR101-GUS, pCambial 300-pHTR102-GUS, pCambial300-pHTR104-GUS and pCambial300-pHTR113-GUS.
将构建的植物表达载体 pCambial300-pHTR101-GUS、 pCambial300-pHTR102-GUS、 pCambial 300-pHTR104-GUS 及 pCambial300-pHTR113-GUS 转 化 农 杆 菌 GyJiOi (Invitrogen) , 筛选阳性克隆。 以获得的含有目的基因质粒的农杆菌转染拟南芥花 序， 用保鲜膜包裹拟南芥苗后暗培养 2天， 之后转入光培养， 在温室培养约 1个半月后 收集种子。 在筛选培养基上筛选 T0代种子， 鉴定 T1代苗后转入盆中， 在温室培养 2周 后， 取叶片进行组织化学染色检测 Gt/S基因的瞬时表达， 如图 13所示。 结果说明玉米 H3.3基因 HTR101 (SEQ ID NO: 13)、 HTR102(SEQ ID NO: 14)、 HTR104(SEQ ID NO: 15)、 和 HrR _?(SEQ ID NO: 16)启动子均具有驱动活性， 且在拟南芥中表达强度从高到低依 次是 HTR10 SEQ ID NO: 15)、 HTR101 (SEQ ID NO: 13)、 HTR102(SEQ ID NO: 14)、 HTR113(SEQ ID NO: 16)。 实施例 8、 转基因拟南芥 GUS组织化学染色检测水稻启动子活性  The plant expression vectors pCambial300-pHTR101-GUS, pCambial300-pHTR102-GUS, pCambial 300-pHTR104-GUS and pCambial300-pHTR113-GUS were transformed into Agrobacterium tumefaciens GyJiOi (Invitrogen), and positive clones were selected. The obtained Agrobacterium containing the plasmid of the target gene was transfected into the Arabidopsis thaliana, and the Arabidopsis thaliana seedlings were wrapped in plastic wrap for 2 days, then transferred to light culture, and the seeds were collected after about one and a half months in the greenhouse. The T0 generation seeds were screened on the screening medium, and the T1 generation seedlings were identified and transferred to the pots. After 2 weeks of greenhouse culture, the leaves were subjected to histochemical staining to detect the transient expression of the Gt/S gene, as shown in Fig. 13. The results indicate that the maize H3.3 gene HTR101 (SEQ ID NO: 13), HTR102 (SEQ ID NO: 14), HTR104 (SEQ ID NO: 15), and HrR _? (SEQ ID NO: 16) promoters are all driven. Activity, and the order of expression in Arabidopsis from high to low is HTR10 SEQ ID NO: 15), HTR101 (SEQ ID NO: 13), HTR102 (SEQ ID NO: 14), HTR113 (SEQ ID NO: 16) . Example 8. Transgenic Arabidopsis thaliana GUS histochemical staining for detection of rice promoter activity
在水稻 Η3.1基因 HrR70< 启动子 (SEQ ID NO: 17)的驱动下， 以 Gt/S为报告基因构 建植物表达载体 pHTR706-GUS。  The plant expression vector pHTR706-GUS was constructed using Gt/S as a reporter gene driven by the rice Η3.1 gene HrR70< promoter (SEQ ID NO: 17).
水稻基因 HTR 70( 启动子序列分别***到 pCambia 1300表达载体 (购自 Cambia公司） 的 Hindlll 禾 B amHI 酶切位点中（即位于 GUS 报告基因的上游）。 GUS 元件***到 PCambial 300 的 BamHI 和 Sacl 酶切位点 。 获得 的重组载体命名 为 ： pCambial 300-pHTR706-GUS。 将 构 建 的 植 物 表 达 载 体 pCambial 300-pHTR706-GUS 转 化 农 杆 菌 GyJiOi(Invitrogen) , 筛选阳性克隆。 以获得的含有目的基因质粒的农杆菌转染拟南芥花 序， 用保鲜膜包裹拟南芥苗后暗培养 2天， 之后转入光培养， 在温室培养约 1个半月后 收集种子。 在筛选培养基上筛选 TO 代种子， 鉴定 T1 代苗后转入盆中， pCambial 300-pHTR706-GUS 的转基因拟南芥在温室培养 2周后， 取叶片进行组织化学 染色检测 Gt/S基因的瞬时表达， 如图 14所示。 结果说明， 水稻 H3.1基因 HrR7(½启动 子 (SEQ ID NO: 17)具有驱动活性， 在拟南芥中较高的表达。 目前来源于玉米 Η3.3基因 HTR101、 HTR102、 HTR104和 HTR113启动子以及来源 于水稻 Η3.3基因 HTR711和 H3.1基因 HTR706验证了它们在拟南芥中有增强表达的效 果， 虽未在水稻和玉米中验证其功能， 但是鉴于它们与来源于拟南芥 H3.3 基因 HTR4 具有高度的同源性， 所以可以理解， 它们在各自相应的作物中也具有很高的表达水平。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被单独引 用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员 可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所附权利要求书所限定 的范围。 The rice gene HTR 70 (promoter sequence was inserted into the Hindlll and BrahHI restriction sites of the pCambia 1300 expression vector (purchased from Cambia), respectively (ie located upstream of the GUS reporter gene). The GUS element was inserted into the BamHI of P Cambial 300. And Sacl cleavage site. The obtained recombinant vector was named: pCambial 300-pHTR706-GUS. The constructed plant expression vector pCambial 300-pHTR706-GUS was transformed into Agrobacterium GyJiOi (Invitrogen), and positive clones were selected. The obtained Agrobacterium containing the plasmid of the target gene was transfected into the Arabidopsis inflorescence, and the Arabidopsis thaliana seedlings were wrapped in plastic wrap for 2 days, then transferred to light culture, and the seeds were collected after about one and a half months in the greenhouse culture. The TO seeds were screened on the screening medium, and the T1 generation seedlings were identified and transferred to the pots. The transgenic Arabidopsis thaliana pCambial 300-pHTR706-GUS was cultured for 2 weeks in the greenhouse, and the leaves were subjected to histochemical staining to detect the Gt/S gene. Transient expression, as shown in Figure 14. The results indicated that the rice H3.1 gene HrR7 (1⁄2 promoter (SEQ ID NO: 17) has a driving activity and is highly expressed in Arabidopsis. Currently derived from the maize Η3.3 genes HTR101, HTR102, HTR104 and HTR113 And the HTR711 and H3.1 genes derived from the rice Η3.3 gene verified that they have enhanced expression in Arabidopsis, although their function was not verified in rice and maize, but they are derived from Arabidopsis thaliana. The H3.3 gene HTR4 has a high degree of homology, so it is understood that they also have high expression levels in their respective crops. All references mentioned in the present application are incorporated herein by reference. Each of the documents is individually referred to as a reference. It is to be understood that various modifications and changes may be made by those skilled in the art after the above teachings of the present invention. The scope defined by the claims is defined.

Claims

权 利 要 求 Rights request
1. 分离的多核苷酸， 其特征在于， 所述的多核苷酸是： 1. Isolated polynucleotide, characterized in that, the polynucleotide is:
(I) SEQ ID NO: 2所示的核苷酸序列的多核苷酸； (I) The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 2;
(2) SEQ ID NO: 3所示的核苷酸序列的多核苷酸； (2) A polynucleotide with the nucleotide sequence shown in SEQ ID NO: 3;
(3) SEQ ID NO: 4所示的核苷酸序列的多核苷酸； (3) A polynucleotide with the nucleotide sequence shown in SEQ ID NO: 4;
(4) SEQ ID NO: 34中（1-743)〜1405位所示的核苷酸序列的多核苷酸； (4) The polynucleotide of the nucleotide sequence shown in positions (1-743) to 1405 in SEQ ID NO: 34;
(5) SEQ ID NO: 35中（1-1076)〜 1369位所示的核苷酸序列的多核苷酸； (5) The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 35 (1-1076) ~ 1369;
(6) SEQ ID NO: 13所示的核苷酸序列的多核苷酸； (6) The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 13;
(7) SEQ ID NO: 14所示的核苷酸序列的多核苷酸； (7) The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 14;
(8) SEQ ID NO: 15所示的核苷酸序列的多核苷酸； (8) The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 15;
(9) SEQ ID NO: 16所示的核苷酸序列的多核苷酸； (9) The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 16;
(10) SEQ ID NO: 17所示的核苷酸序列的多核苷酸； (10) The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 17;
(I I) 核苷酸序列在严格条件下能够与 (1)-(10)任一限定的多核苷酸序列杂交且具有 (1)-(10)任一限定的多核苷酸功能的多核苷酸； 或 (II) A polynucleotide whose nucleotide sequence is capable of hybridizing to any of the polynucleotide sequences defined in (1)-(10) under stringent conditions and has the function of any of the polynucleotides defined in (1)-(10) ; or
(12) 核苷酸序列与（1)-(10)任一限定的多核苷酸序列有 80%以上相同性且具有 (1)-(10)任一限定的多核苷酸功能的多核苷酸。 (12) A polynucleotide whose nucleotide sequence is more than 80% identical to the polynucleotide sequence defined in any one of (1)-(10) and has the function of a polynucleotide defined in any one of (1)-(10) .
2. 如权利要求 1所述的多核苷酸， 其特征在于， （4)中， 包括： 2. The polynucleotide of claim 1, wherein (4) includes:
SEQ ID NO: 34中第 464-1405位 (pHTR4-5(942bp))所示的核苷酸序列的多核苷酸； The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 34 at positions 464-1405 (pHTR4-5 (942bp));
SEQ ID NO: 34中第 276-1405位 (pHTR4-6(1130bp))所示的核苷酸序列的多核苷酸； 或 The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 34 at positions 276-1405 (pHTR4-6 (1130bp)); or
SEQ ID NO: 34所示的核苷酸序列的多核苷酸。 3. 如权利要求 1所述的多核苷酸， 其特征在于， （5)中， 包括： The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 34. 3. The polynucleotide of claim 1, wherein (5) includes:
SEQ ID NO: 3中第 201-521位 (pHTR711-3(521bp))所示的核苷酸序列的多核苷酸； SEQ ID NO: 35 中第 624-1369位 (pHTR711-4(746bp))所示的核苷酸序列的多核苷 酸； Polynucleotide of the nucleotide sequence shown in SEQ ID NO: 3 at positions 201-521 (pHTR711-3 (521bp)); SEQ ID NO: 35 at positions 624-1369 (pHTR711-4 (746bp)) A polynucleotide of the nucleotide sequence shown;
SEQ ID NO: 35 中第 416-1369位 (pHTR711-5(954bp))所示的核苷酸序列的多核苷 酸； The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 35 at positions 416-1369 (pHTR711-5 (954bp));
SEQ ID NO: 35中第 200-1369位 (pHTR711-6(1170bp))所示的核苷酸序列的多核苷 酸； 或 The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 35 at positions 200-1369 (pHTR711-6 (1170bp)); or
SEQ ID NO: 35所示的核苷酸序列的多核苷酸。 4. 权利要求 1所述的多核苷酸的用途， 用于作为启动子元件。 The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 35. 4. Use of the polynucleotide of claim 1 as a promoter element.
5. 如权利要求 4 所述的用途， 其特征在于， 所述的启动子元件用于指导目的基因 强表达。 6. 一种载体， 其特征在于， 所述的载体含有权利要求 1 所述的多核苷酸， 作为启 动子元件。 5. The use as claimed in claim 4, characterized in that the promoter element is used to guide strong expression of the target gene. 6. A vector, characterized in that the vector contains the polynucleotide of claim 1 as a promoter element.
7. 如权利要求 6 所述的载体， 其特征在于， 所述的载体还含有与所述的多核苷酸 操作性连接的目的基因。 7. The vector of claim 6, wherein the vector further contains a target gene operably linked to the polynucleotide.
8. 如权利要求 7 所述的载体， 其特征在于， 所述的目的基因位于所述多核苷酸的 下游。 8. The vector of claim 7, wherein the target gene is located downstream of the polynucleotide.
9. 如权利要求 8 所述的载体， 其特征在于， 所述的目的基因位于所述多核苷酸的 下游， 且与所述多核苷酸的间隔小于 100bp， 更佳地小于 50bp。 9. The vector of claim 8, wherein the target gene is located downstream of the polynucleotide, and the distance from the polynucleotide is less than 100 bp, preferably less than 50 bp.
10. 如权利要求 8所述的载体， 其特征在于， 所述的载体是真核表达载体。 10. The vector according to claim 8, characterized in that the vector is a eukaryotic expression vector.
11. 如权利要求 8所述的载体， 其特征在于， 在所述多核苷酸的下游包含多克隆位 点或至少一个酶切位点。 11. The vector according to claim 8, characterized in that, downstream of the polynucleotide, a multiple cloning site or at least one restriction site is included.
12. 一种遗传工程化的宿主细胞， 其特征在于， 所述的细胞： 12. A genetically engineered host cell, characterized in that the cell:
含有权利要求 6-11任一所述的载体； 或 Containing the carrier described in any one of claims 6-11; or
其基因组中整合有外源的权利要求 1所述的多核苷酸。 The exogenous polynucleotide of claim 1 is integrated into its genome.
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