Application of rice OsWRKY15 gene and encoded protein thereof in regulation and control of drought stress resistance of plants

Abstract

The invention discloses an application of a rice OsWRKY15 gene and an encoding protein thereof in regulating and controlling drought stress resistance of plants. A knockout plant and an overexpression plant of the OsWRKY15 gene are respectively constructed, phenotypes of wild rice, the knockout plant of the OsWRKY15 gene and the overexpression plant of the OsWRKY15 gene are observed under drought treatment, and results show that compared with the wild rice, the wilting and withering degree of the knockout plant of the OsWRKY15 gene is larger than that of the wild rice, and the survival rate of the knockout plant of the OsWRKY15 gene is smaller than that of the wild rice; the wilting and withering degree of an overexpressed plant of the OsWRKY15 gene is smaller than that of wild type rice, and the survival rate of the overexpressed plant is larger than that of the wild type rice. The invention proves that the OsWRKY15 gene has the capability of positively regulating the drought stress resistance of rice, and has application prospects in the aspects of improving the drought stress resistance of plants, cultivating drought stress resistant plant varieties and the like.

Description

Technical Field

[0001] The present invention relates to a drought stress resistance gene isolated from rice and its use, and in particular to a drought stress resistance gene isolated from rice. OsWRKY15 Use of genes and their encoded proteins in regulating plant resistance to drought stress, belonging to OsWRKY15 Genes and their areas of use. Background Art

[0002] In recent years, due to climate change and water shortage, the area of ​​crops affected by drought has continued to expand, seriously threatening food security (HUANG J, YU H, GUAN X, et al, 2016. Accelerated dryland expansion under climate change [J]. Nature Climate Change, 6(2): 166-172). Oryza sativa L.) is the staple food of nearly half of the world's population and is more susceptible to drought stress than other cereal crops (Kondo M., Murty MVR, Aragones DV (2000). Characteristics of root growth and water uptake from soil in upland rice and maize under water stress[J]. Soil Science and Plant Nutrition, 46: 721-732). Rice has a large demand for water, accounting for about 80% of agricultural water use, which leads to many challenges in production when there is a lack of water or insufficient rainfall during the rice growing season. Drought has become one of the main abiotic stress factors affecting rice yield and quality, seriously affecting food security. Therefore, exploring new drought-resistant genes, exploring the molecular mechanism of drought resistance, and breeding drought-resistant rice varieties have become one of the important research contents in the field of rice breeding. Summary of the invention

[0003] The main purpose of the present invention is to OsWRKY15 Gene, OsWRKY15 protein, containing OsWRKY15 Gene expression cassette or containing OsWRKY15 The recombinant plant expression vector of the gene is used to regulate plant resistance to drought stress or cultivate plant varieties resistant to drought stress.

[0004] In order to achieve the above object, the main technical solutions adopted by the present invention include: One aspect of the present invention is to OsWRKY15 Gene, OsWRKY15 protein, containing OsWRKY15Gene expression cassette or containing OsWRKY15 The recombinant plant expression vector of the gene is used to regulate the resistance of plants to drought stress.

[0005] In a preferred embodiment of the present invention, the plants include crops, vegetables, ornamental plants or fruit trees.

[0006] In a preferred embodiment of the present invention, the crops include rice, cotton, corn, sorghum, wheat, soybean, potato, barley, tomato, kidney bean, peanut or sugarcane.

[0007] In a preferred specific embodiment of the present invention, the regulation of plant resistance to drought stress is to improve the resistance of plants to drought stress; wherein, the improvement of plant resistance to drought stress is to reduce the degree of wilting or withering of plants under drought stress or to improve the survival rate of plants under drought stress.

[0008] For reference, the present invention provides an embodiment, that is, by overexpressing the coding gene of the OsWRKY15 protein related to plant drought stress resistance in plants, the expression amount or activity of the OsWRKY15 protein related to plant drought stress resistance is increased, thereby improving the resistance of plants to drought stress, and further improving the survival rate of plants under drought stress.

[0009] A preferred embodiment of the present invention is a method for cultivating a plant variety resistant to drought stress, comprising: overexpressing in a plant OsWRKY15 Gene, OsWRKY15 The expression of the gene is increased or the function or activity of the OsWRKY15 protein is enhanced; for example, OsWRKY15 The gene is connected to the expression control element to obtain a recombinant plant expression vector for expressing the gene in plants; the recombinant plant expression vector is transformed into plants, so that OsWRKY15 The gene is overexpressed in plants, and the resulting transgenic plants have improved resistance to drought stress.

[0010] For reference, the present invention provides a OsWRKY15 Gene plant recombinant expression vector, comprising: OsWRKY15The gene is connected to the expression control element to obtain a recombinant plant expression vector; the recombinant plant expression vector may be composed of a 5' non-coding region, the nucleotides shown in SEQ ID NO.1 and a 3' non-coding region; wherein the 5' non-coding region may include a promoter sequence, an enhancer sequence and / or a translation enhancement sequence; the promoter may be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter; the 3' non-coding region may include a terminator sequence, an mRNA cleavage sequence, etc. Suitable terminator sequences may be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase terminator regions.

[0011] The recombinant plant expression vector may also contain a selective marker gene for selecting transformed cells, for selecting transformed cells or tissues. The marker gene includes genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds. In addition, the marker gene also includes phenotypic markers, such as β-galactosidase and fluorescent protein.

[0012] The transformation protocol and the protocol for introducing the polynucleotide or polypeptide into the plant may vary depending on the type of plant or plant cell being transformed. Suitable methods for introducing the polynucleotide into plant cells include microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-velocity ballistic bombardment. In certain embodiments, various transient transformation methods may be used to introduce the polynucleotide into rice. OsWRKY15 Genes are provided to plants. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0013] A preferred embodiment of the present invention is a method for reducing the resistance of rice to drought stress, comprising: OsWRKY15 Gene mutation OsWRKY15 Reduced gene expression may cause defects in the normal function of OsWRKY15 protein.

[0014] The mutations include OsWRKY15 One or more nucleotides are substituted, deleted and / or added to the nucleotide sequence of a gene or its promoter. Preferably, the mutation can be obtained by physical mutagenesis, chemical mutagenesis, or gene editing. Physical mutagenesis includes, but is not limited to, radiation mutagenesis, space breeding, etc.; chemical mutagenesis includes mutagenesis caused by treatment with mutagens such as EMS; the gene editing method includes, but is not limited to, ZFN, TALE and / or CRISPR / Cas, etc.

[0015] Those skilled in the art can use conventional methods such as conventional gene knockout or gene editing technology to modify the gene in plants. OsWRKY15 Gene knockout mutation, such as constructing OsWRKY15 Gene knockout vectors or constructed using gene editing technology OsWRKY15 CRISPR / Cas9 gene editing vectors, etc., can be used to edit genes in plants OsWRKY15 Gene knockout or mutation are methods that are well known to those skilled in the art.

[0016] Those skilled in the art know that the main principle of the CRISPR / Cas gene editing system or gene editing method is to find the location of gene editing in the host genome through a nucleic acid fragment called guide RNA (gRNA), that is, the targeted DNA sequence, and then cut the DNA through the Cas protein. In this application, the Cas protein includes but is not limited to Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13 and / or Cas14.

[0017] The interference OsWRKY15 The normal expression or normal function of the gene or promoter can be interfered with by RNA interference technology (RNAi) to interfere with the normal expression of the gene encoding the OsWRKY15 protein or its promoter or to cause defects in its normal function. The RNA interference technology is a conventional technology in the art. It specifically binds to the homologous region of the mRNA expressed by the target gene through a 21-23bp short double-stranded RNA (siRNA) or a long double-stranded RNA (dsRNA; double-strand RNA), thereby degrading the mRNA and achieving the effect of inhibiting gene expression.

[0018] Another aspect of the present invention is to provide a rice-derived protein capable of regulating plant resistance to drought stress. OsWRKY15 Genes and their encoded proteins.

[0019] The present invention OsWRKY15 The nucleotide sequence of the gene is selected from the polynucleotide sequences shown in any one of the following (a)-(e): (a) the polynucleotide sequence shown in SEQ ID NO.1; (b) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2; (c) a polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and the polynucleotide sequence still has the function of regulating plant resistance to drought stress; (d) a polynucleotide sequence having at least 95% or more identity with the polynucleotide sequence shown in any one of (a) to (c), and the polynucleotide sequence still has the function of regulating plant resistance to drought stress; (e) A polynucleotide sequence that is complementary to the polynucleotide sequence described in any one of (a) to (d), and the polynucleotide sequence still has the function of regulating plant resistance to drought stress.

[0020] The percentage of sequence identity described in the present invention can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0021] In addition, those skilled in the art can also optimize the nucleotide sequence shown in SEQ ID NO. 1 to enhance the expression efficiency in plants.

[0022] The present invention can also be carried out by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID NO.1, and / or performing missense mutation of one or several base pairs.

[0023] Those skilled in the art can easily adopt known methods, such as directed evolution or point mutation, to OsWRKY15 The nucleotide sequence of the gene is mutated. OsWRKY15 Nucleotides with 75% or higher identity to the nucleotide sequence of a gene are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the encoded protein has the function of regulating plant resistance to drought stress.

[0024] In addition, the nucleotide sequence described in the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; it can also be RNA, such as mRNA or hnRNA.

[0025] The amino acid sequence of the OsWRKY15 protein of the present invention is selected from any one of the following amino acid sequences (a) to (d): (a) the amino acid sequence shown in SEQ ID NO.2; (b) a protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2, wherein the protein variant still has the function or activity of regulating plant resistance to drought stress; (c) a protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.2, wherein the protein variant still has the function or activity of regulating plant resistance to drought stress; (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 2, wherein the protein still has the function or activity of regulating plant resistance to drought stress.

[0026] In the early stage, we conducted genome-wide association analysis and drought QTL positioning on 411 experimental materials planted in water-dry fields, combined with the results of drought treatment expression level analysis, and screened out OsWRKY15 The present invention firstly uses 20% PEG6000 to simulate drought stress or ABA to treat rice plants, and finds that OsWRKY15 The relative expression of the gene increases with the extension of the treatment time. OsWRKY15 Gene knockout plants and OsWRKY15 gene overexpression plants and observed the wild-type rice, OsWRKY15 The phenotypes of gene knockout plants and overexpression plants were compared with wild-type rice. OsWRKY15 The knockout plants wilted and withered more than wild-type rice, and had a lower survival rate than wild-type rice; OsWRKY15 The degree of wilting and withering of the gene-overexpressing plants was less than that of wild-type rice, and the survival rate was higher than that of wild-type rice; OsWRKY15 The gene has the ability to regulate rice's resistance to drought stress. The invention has application prospects in improving plants' resistance to drought stress, and the invention can be used to cultivate drought-resistant rice varieties and apply them to actual production processes.

[0027] Definitions of terms used in this invention Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs (phosphorothioates, phosphoramidates, etc.) used in antisense technology. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0029] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, descriptions directed to polypeptides apply equally to describing peptides and describing proteins, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

[0030] The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f factor, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

[0031] The term "operably linked" refers to a functional connection between two or more elements. The operably linked elements may be contiguous or non-contiguous.

[0032] The term "recombinant plant expression vector" means one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, together with vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selection markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc.

[0033] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0034] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 for OsWRKY15 The relative expression results of genes after 20% PEG6000 simulated drought stress treatment or ABA treatment; Figure 1 - (a) is OsWRKY15 The detection results of relative expression of genes after 20% PEG6000 simulated drought stress treatment; Figure 1 - (b) OsWRKY15 Detection results of relative expression levels of genes after ABA treatment.

[0036] Figure 2For rice OsWRKY15 The results of identification of genetically modified strains; among them, Figure 2 - (a) is OsWRKY15 Knockout strain sequences; Figure 2 - (b) shows the results of Sanger sequencing wrky15-1 Strains and wrky15-2 in strains OsWRKY15 The DNA sequence of the gene has single-base deletions and single-base insertions; Figure 2 - (c) Rice OsWRKY15 PCR positive identification of gene overexpression strains; among them, lane 1 is a 5000bp Marker, lanes 2-7 are the identification results of the overexpression strain OE, and lanes 8-11 are controls for wild-type plants WT with overexpression identification primers added.

[0037] Figure 3 for OsWRKY15 Phenotypic diagram of gene knockout strains and wild-type plants WT under normal and drought stress; Figure 3 - (a) is OsWRKY15 Phenotypic results of soil drought stress in gene knockout lines; Figure 3 - (b) OsWRKY15 Phenotypic results of 20% PEG6000 stress in the knockout strains; Figure 3 - (c) OsWRKY15 Survival rate of soil drought in knockout lines; Figure 3 - (d) OsWRKY15 Survival rate of gene knockout lines under 20% PEG6000 stress.

[0038] Figure 4 for OsWRKY15 Phenotypic diagram of gene overexpression strains and wild-type plants WT under normal and drought stress; Figure 4 - (a) is OsWRKY15 Phenotypic results of soil drought stress in gene overexpression lines; Figure 4 - (b) OsWRKY15 Survival rate of gene overexpression lines under soil drought stress (OE1); Figure 4 - (c) OsWRKY15 Survival rate of gene overexpression lines under soil drought stress (OE2). DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0040] Test Example 1 OsWRKY15 Gene expression level detection test after 20% PEG6000 simulated drought stress or ABA treatment 1 Test methods 1.1 Sample collection and processing Select the plump and uniform rice Zhonghua 11 as the wild-type material seeds, and place them in a 50°C oven for two days to break dormancy, then sterilize them in a 0.5% sodium hypochlorite solution for 20 minutes. Rinse them again with tap water for 3 times, soak them in water, and then place them in an artificial climate room at 28°C for germination for about 3 days. When the seeds germinate 2-3 mm in hydroponics, transfer them to the PCR plate with the cut bottom to float on tap water (pH 5.0-5.5) for growth. After one week, transfer them to Yoshida culture medium (Coolaber, NSP1040) for culture, and adjust the pH to 5.0-5.5. The culture medium is changed every Monday. 20% PEG6000 or ABA was added to the culture medium of seedlings grown to the three-leaf stage, and the aboveground parts were sampled after 0 h, 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h and 48 h of simulated drought stress treatment. Five plants were taken each time, and the materials were quick-frozen in liquid nitrogen and stored in a -80°C refrigerator for later use.

[0041] 1.2 Extraction of total RNA from rice and RT-PCR detection 1.2.1 Extraction of total RNA from plant tissues (1) Grind the plant tissue samples frozen in liquid nitrogen into powder and dispense them into RNase-free tubes at a rate of about 150 mg per tube. Add 1 mL of TRIzol to each tube of experimental sample, shake thoroughly for 5 min, and place on ice for 5 min.

[0042] (2) Add 200 μL of chloroform to each tube for extraction, shake thoroughly for 2 min, and place on ice for 2 min.

[0043] (3) Centrifuge at 12,000 g, 4°C for 10 min and transfer 400 μL of the supernatant into a new RNase-free tube.

[0044] (4) Add 400 μL of isopropanol to each tube, mix well, and place in a -20°C refrigerator for 2 h.

[0045] (5) Centrifuge at 12,000 g for 10 min at 4°C, discard the supernatant, and add 1 mL of pre-cooled 75% ethanol to suspend the precipitate; centrifuge at 12,000 g for 3 min at 4°C, discard the supernatant, and repeat twice.

[0046] (6) Place the centrifuge tube on ice and blow dry it in a clean bench for 10 min.

[0047] (7) Dissolve the precipitate in 30 μL DEPC water in each tube. The precipitate can be used for RNA reverse transcription or stored in a -80°C refrigerator for later use.

[0048] 1.2.2 Reverse transcription of RNA into cDNA (1) Determine RNA concentration using Nanodrop and add 1 μg RNA to the reverse transcription system based on the concentration.

[0049] (2) The amount of reverse transcription reagent (ABclonal, China) added to each system: 4 μL 5×ABScript III RTMix, 1 μL 20×gDNA Remover Mix, and Nuclease-free H2O to 20 μL.

[0050] (3) Perform reverse transcription reaction on a PCR instrument according to the following reaction program: 37℃ for 2 min, 55℃ for 15 min, 85℃ for 5 min, and hold at 4℃.

[0051] (4) Determine the cDNA concentration by Nanodrop and store at -20°C until needed.

[0052] 1.2.3 Real-time quantitative PCR (RT-qPCR) The nucleotide sequences of the primers used in real-time fluorescence quantification are as follows: OsWRKY15 -RT-F:GAGCAAGCAGGTGAAGGTGA (SEQ ID NO.4); OsWRKY15 -RT-R:TGGTACACGACGTCGAAGAG (SEQ ID NO. 5).

[0053] QUR -RT-F:GCTCCGTGGCGGTATCAT (SEQ ID NO.6); QUR -RT-R:CGGCAGTTGACAGCCCTAG (SEQ ID NO. 7).

[0054] RT-PCR was performed using a real-time fluorescence PCR instrument according to the following reaction system (Table 1) and operating program (Table 2); the ΔΔCt method (Livak, KJ, and Schmittgen, TD (2001) was used to analyze the relative gene expression data. −ΔΔCT Method. Methods. 25, 402–408) calculation OsWRKY15 Gene relative to QUR Gene expression level.

[0055] Table 1 RT-qPCR reaction system

[0056] Table 2 RT-qPCR operation procedures

[0057] 2 Test results OsWRKY15 The expression level changes of genes after 20% PEG6000 simulated drought stress or ABA treatment are as follows Figure 1 As shown, through detection OsWRKY15 The changes in gene expression with the extension of treatment time showed that when treated with 20% PEG6000 ( Figure 1 -(a)) or ABA treatment ( Figure 1 - (b) After OsWRKY15 The relative expression of the gene increased, that is, OsWRKY15 Gene expression is upregulated by drought, indicating OsWRKY15 Genes involved in rice response to drought stress.

[0058] Test Example 2 OsWRKY15 Genetic transformation and identification experiment in rice 1 OsWRKY15 Gene CRISPR knockout vector construction according to OsWRKY15 The target site for knockout was designed based on the gene cDNA sequence, and the website used was: http: / / skl.scau.edu.cn / dsdecode / .

[0059] The nucleotide sequence of the sgRNA target site is: ACCGGTCTATAAGCATCGCGAGG (SEQ ID NO. 8).

[0060] The experimental methods and vectors were based on the literature (Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants. Molecular Plant, 2015, 8(8): 1274-1284), and the recombinant vector was named pYLCRISPR / Cas9Pubi-OsWRKY15.

[0061] OsWRKY15The nucleotide sequence of the CDS of the gene is shown in SEQ ID NO.1: ATGGCCGCCGGAGCGGACGAGCGGTGCCGCGCGCTGGTGTCCGGCCTGCTGTCGTCGATCGACCGGTCTATAAGCATCGCGAGGTCGTGCTGCACGGAGGCCGCCGCCGCCGGGCGCCTGACCCAGCAGGCCGGCGCCGCGCCTGAGTCGCCGCCGTCCGCCGACGGTAGCGCCGGCTCGGACCTCGGCGCCGACTCCCGGTGCCGCGCCAATGCGGCGGGGCCGTGCAAGAAGAGGAAGACGCTGCCCAAGTGGAGCAAGCAGGTGAAGGTGAGGTCCGTGCAGGACGTCGGCCCCCTCGACGACGGGTTCAGCTGGAGGAAGTACGGGCAGAAGGACATCCTCGGCGCCAAGTACCCAAGAGCCTACTTCCGGTGCACGCACCGGCACACGCAGGGCTGCCACGCCAGCAAGCAGGTGCAGCGCGCGGACGGCGACCCGCTGCTCTTCGACGTCGTGTACCACGGCGACCACACCTGCGCGCATGGCGTGCGCTCCGCCGCCGCCGCCATCGACGGCCAGGCCGCGGCGAGCGCGGAGCAAAAGCATCAGCCGACGCCGCCGCAGGAGCAGAACGCCGTCTCGGTCGCGTTCACCTCCATGGCCGTCGTCAACGCATCGACGTCGTCGCCCTTCGTGTCCCCGGCGATGTCGGACTGCCAGATTAGCTACGAGCTGGGCGGTGGCAGCATGGCCGGAGTCCGCAACGTGCCCGACGTGGAGCTTGCCTCCAAGACGAACTCCTCCATGGGAGACGACATGGAGTTCATGTTCTCGCTGGACTCCGATTTCTTGGACACCTACAAGTACTCCAGCTATTTCTAA (SEQ ID NO.1).

[0062] The amino acid sequence of the OsWRKY15 protein is shown in SEQ ID NO.2: MAAGADERCRALVSGLLSSIDRSISIARSCCTEAAAAGRLTQQAGAAPESPPSADGSAGSDLGADSRCRANAAGPCKKRKTLPKWSKQVKVRSVQDVGPLDDGFSWRKYGQKDILGAKYPRAYFRCTHRHTQGCHASKQVQRADGDPLLFDVVYHGDHTCAHGVRSAAAAIDGQAAASAEQKHQPTPPQEQNAVSVAFTSMAVVNASTSSPFVSPAMSDCQISYELGGGSMAGVRNVPDVELASKTNSSMGDDMEFMFSLDSDFLDTYKYSSYF (SEQ ID NO.2).

[0063] OsWRKY15

[0064] 2 OsWRKY15 Gene overexpression vector construction The overexpression vector was constructed by the company and the recombinant vector was named pBWA(V)HS-OsWRKY15.

[0065] 3 Agrobacterium transformation The knockout vector pYLCRISPR / Cas9Pubi-OsWRKY15 and the overexpression vector pBWA(V)HS-OsWRKY15 were transformed into Agrobacterium EHA105 competent cells by freeze-thaw method (Ruifang Yang ,Qicai Tang ,HuimeiWang,Xiaobo Zhang ,Gang Pan ,HongWang and Jumin Tu Analyses of two rice( Oryza sativa )cyclin-dependent kinase inhibitors and effects of transgenic expression of OsiICK6 on plant growth and development, 2011, Annals of Botany, 107:1087-1101), and the method was referred to the molecular cloning experimental guide.

[0066] 4 Genetic transformation Genetic transformation was carried out using Agrobacterium-mediated genetic transformation method with rice Zhonghua 11 as the recipient material. The culture medium formula is shown in Table 3.

[0067] Table 3 Culture medium and its formula used for genetic transformation

[0068] The specific method is as follows: 1. Callus induction Take an appropriate amount of mature rice seeds, remove the shells, wash and disinfect with 70% alcohol for 1 min, shake continuously, and then disinfect with 15% sodium hypochlorite for 30 min (can be placed on a shaker); finally rinse with sterile distilled water 4-5 times, use sterile filter paper to dry the surface moisture of the seeds and then inoculate. Inoculate the disinfected seeds in an induction medium containing 2.0 mg / L 2,4-D and culture in the dark at 28℃ for 30-40 days. The callus obtained by culture is expanded on the subculture medium and subcultured every 2 weeks until the embryonic callus is formed.

[0069] 2. Agrobacterium infection (1) Agrobacterium carrying the knockout vector pYLCRISPR / Cas9Pubi-OsWRKY15 and the overexpression vector pBWA(V)HS-OsWRKY15 were streaked onto the surface of LB solid culture medium containing antibiotics (50 mg / L kanamycin and 25 mg / L rifampicin) and cultured overnight at 28°C and 200 rpm.

[0070] (2) Use a sterilized toothpick to pick a single clone and inoculate it into 5 mL of LB liquid medium (Coolaber, PM0010) containing antibiotics (50 mg / L kanamycin and 25 mg / L rifampicin). Cultivate at 28°C with shaking until the OD reaches 0. 600 =0.5.

[0071] (3) Take the activated fresh bacterial solution and inoculate it into 25 mL of the same LB liquid medium at a ratio of 1:100, and culture it under the same conditions until the OD 600 =0.5.

[0072] (4) Centrifuge the bacterial solution at 5000 g, 4°C for 10 min to collect the cells and discard the supernatant. Add 25 mL of 10 mM MgSO4 to suspend the cells and gently pipette to fully suspend them. Centrifuge the solution at 5000 g, 4°C for 10 min to re-collect the cells and discard the supernatant.

[0073] (5) Resuspend in 25 mL of infection medium (AA-AS) containing 200 μM acetosyringone (AS).

[0074] (6) Transfer the embryonic callus tissue with good growth status from the subculture medium to a culture dish covered with sterile filter paper (cut the callus tissue into 0.3-0.4 mm size) and air-dry it on a clean bench for 10-20 min.

[0075] (7) Immerse the dried embryonic callus in a 50 mL centrifuge tube containing the above bacterial solution for 20 min, shaking it every 5 min. Pour off the bacterial solution, take out the callus and place it on sterile filter paper to air-dry for 10-20 min, then transfer it to co-cultivation medium (CC) containing 200 μM acetosyringone (AS) covered with sterile filter paper, and co-cultivate it at 25°C in the dark for 3 days.

[0076] (8) Collect the callus tissue with no obvious Agrobacterium on the surface, rinse it three times with sterile water containing 600 mg / L cephalosporin, and absorb the excess water.

[0077] (9) The callus tissue was transferred to the screening medium (N6 medium containing 500 mg / L cephalosporin and 50 mg / L hygromycin) and the screening was continued for 2-3 times, each time for two weeks. Finally, well-growing bright yellow hygromycin-resistant callus tissue was obtained.

[0078] 3. Regeneration of transformant plants Take fresh hygromycin-resistant callus, cut the callus into 2 mm small pieces, inoculate in pre-regeneration medium, culture in dark at 28℃ for 7 days, then place in light culture room (12 h light / 12 h dark) for 8-9 days, transfer the callus that has differentiated adventitious buds to regeneration medium (250 mL tissue culture bottle), and continue light culture. Wait until the adventitious buds grow into 4-6 cm high seedlings and then transfer to rooting medium, culture in light culture room (12 h light / 12 h dark) at 28℃ for about 15 days, obtain transformant plants, move to greenhouse for planting (T0 generation), take leaves one month later for PCR positive identification using primers Hyg-F / R, and harvest positive transgenic plants (T1 generation) from positive plants.

[0079] The nucleotide sequence of primer Hyg-F / R is as follows: Hyg-F: CAAAGATCGTTATGTTTATCGGCACT (SEQ ID NO.9); Hyg-R:TTGGCGACCTCGTATTGGGAA (SEQ ID NO. 10).

[0080] 5TPS method for extracting rice genomic DNA (1) Place plant leaves in 2 mL centrifuge tubes, add 5 mm diameter steel beads to each tube, freeze quickly in liquid nitrogen, and grind the samples with a tissue grinder at 1500 rpm for about 90 s; (2) Add 1000 μL of TPS extract to each tube, mix thoroughly, and place in a 65°C oven for 30 min, inverting twice in the middle to mix thoroughly; (3) Centrifuge at 12,000 g for 10 min at room temperature and transfer approximately 500 μL of supernatant into a new 1.5 mL centrifuge tube; (4) Add 400 μL of isopropanol and mix gently, then let stand at room temperature for 10 min. (5) Centrifuge at 12,000 g for 10 min at room temperature, discard the supernatant, add 1 mL of 70% ethanol to suspend the precipitate; discard the supernatant, add 1 mL of 70% ethanol to suspend the precipitate, and discard the supernatant; (6) Blow dry in a clean bench, add 50 μL ddH2O to dissolve the DNA, and store at 4°C until used.

[0081] 6 OsWRKY15 Identification of gene knockout rice and analysis of sgRNA target editing Extraction of T2 generation OsWRKY15 The genomic DNA of the gene knockout strain was extracted, and the primers OsWRKY15-KO-F / R were designed with the target site as the center to amplify the sequence. The PCR amplification products were sequenced. By comparing the sequencing results, two different gene knockout materials were obtained. Compared with the nucleotide sequence at the wild-type target site (SEQ ID NO.8), wrky15-1 The strain lacked 1 base at the target site (ACCGGTCTATAAGCATGCGAGG (SEQ ID NO. 13)); wrky15-2 The strain has one base inserted at the target site (ACCGGTCTATAAGCATCAGCGAGG (SEQ ID NO.14)). The deletion or insertion of bases in the above two strains will cause frameshift mutations, resulting in protein sequences without conserved domains, resulting in loss of OsWRKY15 protein function, and two OsWRKY15- KO The strains can be used for subsequent phenotypic observation experiments ( Figure 2 - (a), Figure 2 - (b)).

[0082] The nucleotide sequence of primer OsWRKY15-KO-F / R is as follows: OsWRKY15-KO-F:GAGCGGACGAGCGGTG (SEQ ID NO. 11); OsWRKY15-KO-R:TAGAAACCGGTCAACGTGCC (SEQ ID NO. 12).

[0083] 7 OsWRKY15 Identification of gene overexpression strains Extraction of T2 generation OsWRKY15 The genomic DNA of the gene overexpression strain was amplified with primers OsWRKY15-OE-F / R. The PCR amplification products were run on agarose gels, and the strains with bands were overexpression positive strains. Two OsWRKY15 - OE The strains can be used for subsequent phenotypic observation experiments ( Figure 2 - (c)).

[0084] OsWRKY15-OE-F:AGTGGAAAAGGAAGGTGGCT (SEQ ID NO.15); OsWRKY15-OE-R:CGTCTTCCTCTTCTTGCACG (SEQ ID NO. 16).

[0085] Test Example 3 OsWRKY15 Phenotypic identification test of gene overexpression and knockout lines after drought treatment Phenotypic identification test of 120% PEG6000 simulated drought stress treatment To make it clear OsWRKY15 Tolerance of knockout materials to drought stress. Select plump and uniform seeds of wild type and knockout mutant materials, place them in a 50°C oven for two days to break dormancy, and then sterilize them in 0.5% sodium hypochlorite solution for 20 minutes. Rinse again with tap water three times, soak with water, and then place in an artificial climate room at 28°C for germination for about 3 days. When the seeds germinate 2-3 mm in hydroponics, transfer them to a PCR plate with a cut bottom and float on tap water (pH 5.0-5.5) for growth. After one week, transfer them to Yoshida culture medium and adjust the pH to 5.0-5.5. The culture medium is changed every Monday. 20% PEG6000 is applied to the culture medium of seedlings grown to the three-leaf stage. Three replicates are set for the treatment, with 24 seeds of wild type and knockout materials in each replicate. The phenotype is observed after 14 days of drought stress treatment.

[0086] The test results are as follows Figure 3 As shown, compared with wild-type rice WT, OsWRKY15 Knockout mutant plants are more sensitive to drought stress ( Figure 3 - (b)), OsWRKY15 The survival rate of gene knockout plants was also lower than that of wild type WT ( Figure 3 -(d)), indicating that knockout rice OsWRKY15 Genes can significantly affect rice's ability to resist drought stress.

[0087] 2 Soil drought phenotype identification test To confirm OsWRKY15 Genes positively regulate rice drought resistance. Seeds of overexpression, knockout lines and wild-type WT plants were soaked in water at 28-30°C for 3 days, and the germinated seeds were planted in pots containing a mixture of nutrient soil and vermiculite. The pots were placed in a container of water and grown in the sun for 1 month (three-leaf stage) in summer (25-35°C). Without watering, drought stress was applied to the leaves and stems to wilt (about 7 days), and then re-watered for 7 days. The survival rate of each line was counted. Transgenic lines and control lines were planted in the same pot.

[0088] OsWRKY15 The experimental results of gene knockout strains are as follows Figure 3 As shown, OsWRKY15 The knockout plants wilted and withered more than the wild type WT ( Figure 3 - (a)), OsWRKY15 The survival rate of gene knockout plants was also lower than that of wild type WT ( Figure 3-(c)), thus knocking out mutant rice in rice OsWRKY15 Plants with the gene were more sensitive to drought stress.

[0089] OsWRKY15 The experimental results of gene overexpression strains are as follows Figure 4 As shown, OsWRKY15 The degree of wilt and withering of the gene overexpression plants was less than that of the wild type WT ( Figure 4 - (a)), OsWRKY15 The survival rate of the gene overexpression plants was also higher than that of the wild type WT ( Figure 4 - (b), Figure 4 -(c)), thus overexpressing rice in rice OsWRKY15 The plants are more drought resistant after the gene is added. OsWRKY15 Gene positively regulates rice drought resistance, so rice OsWRKY15 The gene and its encoded protein have the ability to enhance the crops' resistance to drought stress.

Claims

1. OsWRKY15 Gene, OsWRKY15 protein, containing OsWRKY15 Gene expression cassette or containing OsWRKY15 The invention discloses a use of a recombinant plant expression vector of a gene in regulating the resistance of plants to drought stress; the plants include crops, vegetables, ornamental plants or fruit trees.

2. The use according to claim 1, characterized in that The regulation of plant resistance to drought stress is to improve the plant resistance to drought stress.

3. The use according to claim 2, characterized in that: The improvement of the plant's resistance to drought stress is to reduce the degree of wilting or withering of the plant under drought stress or to increase the survival rate of the plant under drought stress.

4. The use according to claim 1, characterized in that The crops include rice, cotton, corn, sorghum, wheat, soybean, potato, barley, tomato, kidney bean, peanut or sugar cane.

5. The use according to any one of claims 1 to 4, characterized in that: include: Will OsWRKY15 Overexpression of genes in plants increases OsWRKY15 The expression amount or expression level of the gene; or enhancing the function or activity of the OsWRKY15 protein.

6. The use according to claim 1, characterized in that: The regulation of plant resistance to drought stress is to reduce the plant's resistance to drought stress.

7. The use according to claim 6, characterized in that include: The rice OsWRKY15 Gene mutation or silencing OsWRKY15 Reduced gene expression may cause defects in the normal function of OsWRKY15 protein.

8. The use according to claim 1, characterized in that The OsWRKY15 The nucleotide sequence of the CDS of the gene is selected from the polynucleotide sequences described in (a) or (b) below: (a) the polynucleotide sequence shown in SEQ ID NO.1; (b) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2; The amino acid sequence of the OsWRKY15 protein is shown in SEQ ID NO.

2.

9. A method for cultivating plant varieties resistant to drought stress, characterized in that: include: Build contains OsWRKY15 Gene overexpression recombinant plant expression vector; transforming the overexpression recombinant plant expression vector into plants, so that OsWRKY15 The gene is overexpressed in plants, and the resulting transgenic plants have improved resistance to drought stress; The OsWRKY15 The nucleotide sequence of the CDS of the gene is selected from the polynucleotide sequences described in (a) or (b) below: (a) the polynucleotide sequence shown in SEQ ID NO.1; (b) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2; The plants include crops, vegetables, ornamental plants or fruit trees.

10. The method according to claim 9, characterized in that The crops include rice, cotton, corn, sorghum, wheat, soybean, potato, barley, tomato, kidney bean, peanut or sugar cane.

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