Use of Rice OsWRKY15 Gene and Its Encoded Protein in Regulating Plant Drought Stress Resistance

Abstract

The present invention discloses the use of a rice OsWRKY15 gene and its encoded protein in regulating plant drought stress resistance. The present invention respectively constructs OsWRKY15 knockout plants and overexpression plants of the gene, and observes the phenotypes of wild-type rice, OsWRKY15 knockout plants and overexpression plants of the gene under drought treatment. The results show that compared with wild-type rice, OsWRKY15 the degree of wilting and withering of the knockout plants of the gene is greater than that of wild-type rice, and the survival rate is less than that of wild-type rice; OsWRKY15 the degree of wilting and withering of the overexpression plants of the gene is less than that of wild-type rice, and the survival rate is greater than that of wild-type rice. The present invention proves that OsWRKY15 the gene has the ability to positively regulate rice drought stress resistance and has application prospects in improving plant drought stress resistance and cultivating plant varieties with drought stress resistance, etc.

Description

Technical Field

[0001] The present invention relates to a gene isolated from rice that resists drought stress and its uses, and particularly relates to the use of a gene isolated from rice and its encoded protein in regulating plant drought stress resistance, belonging to the field of genes and their uses. OsWRKY15 the field of genes and their uses. OsWRKY15 gene and its uses. Background Art

[0002] In recent years, affected by climate change and water resource shortage, the area of crops affected by drought has been continuously expanding, 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). Rice ( Oryza sativa L.) is the staple food for nearly half of the world's population. Compared with other cereal crops, it is more vulnerable to drought stress (Kondo M., Murty M.V.R., Aragones D.V. (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 relatively large water demand, accounting for about 80% of agricultural water use. This 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, clarifying 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 object of the present invention is to apply the OsWRKY15 gene, OsWRKY15 protein, an expression cassette containing the OsWRKY15 gene or a recombinant plant expression vector containing the OsWRKY15 gene to regulating plant drought stress resistance or cultivating plant varieties resistant to drought stress.

[0004] To achieve the above object, the main technical solutions adopted by the present invention include:

[0005] On the one hand, the present invention is to apply the OsWRKY15 gene, OsWRKY15 protein, an expression cassette containingOsWRKY15 The expression cassette of the gene or the recombinant plant expression vector containing OsWRKY15 the gene is applied to regulate the drought stress resistance of plants.

[0006] In a preferred specific embodiment of the present invention, the plants include crops, vegetables, ornamental plants or fruit trees.

[0007] In a preferred specific embodiment of the present invention, the crops include rice, cotton, corn, sorghum, wheat, soybean, potato, barley, tomato, kidney bean, peanut or sugarcane.

[0008] In a preferred specific embodiment of the present invention, the regulation of plant drought stress resistance is to improve the drought stress resistance of plants; wherein, the improvement of plant drought stress resistance is to reduce the wilting or withering degree of plants under drought stress or to increase the survival rate of plants under drought stress.

[0009] As a 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 level 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 increasing the survival rate of plants under drought stress.

[0010] In a preferred specific embodiment of the present invention, a method for cultivating a plant variety with drought stress resistance includes: overexpressing OsWRKY15 the gene in plants to enhance OsWRKY15 the expression level of the gene or to enhance the function or activity of the OsWRKY15 protein; for example, connecting the OsWRKY15 gene derived from rice with an expression regulatory element to obtain a recombinant plant expression vector for expressing the gene in plants; transforming the recombinant plant expression vector into plants to make OsWRKY15 the gene be overexpressed in plants, and the obtained transgenic plants have improved resistance to drought stress.

[0011] As a reference, the present invention provides a OsWRKY15 gene plant recombinant expression vector, including: the OsWRKY15A recombinant plant expression vector is obtained by connecting a gene with expression regulatory elements; the recombinant plant expression vector can be composed of a 5′-untranslated region, the nucleotide shown in SEQ ID NO.1, and a 3′-untranslated region; wherein, the 5′-untranslated region may include a promoter sequence, an enhancer sequence, or / and a translation enhancer sequence; the promoter may be a constitutive promoter, an inducible promoter, a tissue- or organ-specific promoter; the 3′-untranslated region may contain a terminator sequence, an mRNA cleavage sequence, etc. Suitable terminator sequences can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase terminator regions.

[0012] The recombinant plant expression vector may also contain a selectable marker gene for selecting transformed cells, for selecting transformed cells or tissues. The marker genes include: genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds, etc. In addition, the marker genes also include phenotypic markers, such as β-galactosidase and fluorescent proteins, etc.

[0013] The transformation protocol and the protocol for introducing the polynucleotide or polypeptide into a plant may vary depending on the type of plant or plant cell to be transformed. Suitable methods for introducing the polynucleotide into a plant cell include: microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-velocity ballistic bombardment, etc. In a specific embodiment, various transient transformation methods can be used to provide the OsWRKY15 gene to a plant. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0014] A preferred specific embodiment of the present invention, a method for reducing the drought stress resistance of rice, includes: mutating the OsWRKY15 gene in rice to OsWRKY15 reduce the expression level of the gene or cause defects in the normal function of the OsWRKY15 protein.

[0015] The mutation includes one or more nucleotide substitutions, deletions, and / or additions in the OsWRKY15 gene or the nucleotide sequence of 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 methods include mutagenesis caused by treating with mutagens such as EMS, etc.; the gene editing methods include, but are not limited to, ZFN, TALE, and / or CRISPR / Cas, etc.

[0016] Those skilled in the art can use conventional methods such as conventional gene knockout or gene editing techniques toOsWRKY15 The gene is knocked out and mutated. For example, a OsWRKY15 gene knockout vector is constructed or a CRISPR / Cas9 gene editing vector for the OsWRKY15 gene is constructed by using gene editing technology. The OsWRKY15 gene in the plant is knocked out or mutated. These methods are all well-known to those skilled in the art.

[0017] 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 position to be gene-edited in the host genome through a nucleic acid fragment called guide RNA (gRNA), that is, the target DNA sequence, and then cut the DNA by the Cas protein. In this application, the Cas protein includes but is not limited to proteins such as Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13, and / or Cas14.

[0018] The normal expression or normal function of the OsWRKY15 gene or promoter can be interfered with by using 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. RNA interference technology is a conventional technology in this field. 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), degrades the mRNA, and achieves the effect of inhibiting gene expression.

[0019] Another aspect of the present invention is to provide a OsWRKY15 gene derived from rice that can regulate plant drought stress resistance and its encoded protein.

[0020] In the present invention, the OsWRKY15 nucleotide sequence of the gene is selected from any one of the following polynucleotide sequences (a)-(e):

[0021] (a) The polynucleotide sequence shown in SEQ ID NO.1;

[0022] (b) The polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2;

[0023] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and this polynucleotide sequence still has the function of regulating plant drought stress resistance;

[0024] (d) A polynucleotide sequence having at least 95% or more identity with the polynucleotide sequence shown in any one of (a)-(c), and this polynucleotide sequence still has the function of regulating plant drought stress resistance;

[0025] (e) A polynucleotide sequence capable of complementary pairing with the polynucleotide sequence described in any one of (a)-(d), and this polynucleotide sequence still has the function of regulating plant drought stress resistance.

[0026] The percentage of sequence identity described in the present invention can be obtained by well-known bioinformatics algorithms, including the Myers and Miller algorithm, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the algorithm of Karlin and Altschul, which are well-known to those skilled in the art.

[0027] In addition, those skilled in the art can also optimize the nucleotides shown in SEQ ID NO.1 to enhance the expression efficiency in plants.

[0028] The present invention can also be achieved by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO.1, and / or performing a missense mutation of one or several base pairs.

[0029] Those skilled in the art can easily use known methods, such as directed evolution or point mutation methods, to OsWRKY15 mutate the nucleotide sequence of the gene. Those artificially modified nucleotides having 75% or higher identity with the OsWRKY15 nucleotide sequence of the gene, as long as the encoded protein has the function of regulating plant drought stress resistance, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0030] 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, etc.

[0031] The amino acid sequence of the OsWRKY15 protein described in the present invention is selected from the amino acid sequences shown in any one of the following (a)-(d):

[0032] (a) The amino acid sequence shown in SEQ ID NO.2;

[0033] (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, and this protein variant still has the function or activity of regulating plant drought stress resistance;

[0034] (c) A protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.2, and this protein variant still has the function or activity of regulating plant drought stress resistance;

[0035] (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO.2, and this protein still has the function or activity of regulating plant drought stress resistance.

[0036] Previously, through genome-wide association analysis and drought QTL mapping of 411 experimental materials planted in paddy fields and dry fields respectively, combined with the analysis results of expression levels under drought treatment, OsWRKY15 it was screened out that may have the ability to enhance rice's resistance to drought stress. First, the present invention uses 20% PEG6000 to simulate drought stress treatment or ABA treatment on rice plants, and it is found that OsWRKY15 the relative expression level of the gene increases with the extension of the treatment time. The present invention further constructs OsWRKY15 knockout plants of the gene and OsWRKY15 overexpression plants of the gene, and observes the phenotypes of wild-type rice, OsWRKY15 knockout plants and overexpression plants of the gene under drought treatment; the results show that compared with wild-type rice, OsWRKY15 the wilting and withering degrees of the knockout plants of the gene are greater than those of wild-type rice, and the survival rate is less than that of wild-type rice; OsWRKY15 the wilting and withering degrees of the overexpression plants of the gene are less than those of wild-type rice, and the survival rate is greater than that of wild-type rice; it is proved that OsWRKY15 the gene has the ability to regulate rice's resistance to drought stress. The present invention has application prospects in improving the resistance of plants to drought stress, and can use the present invention to cultivate drought-tolerant rice varieties and apply them to the actual production process.

[0037] Term definitions involved in the present invention

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0039] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides in single-stranded or double-stranded form, and polymers thereof. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as the explicitly specified sequence. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed-base and / or deoxyinosine residue.

[0040] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, a description of a polypeptide applies equally to the description of a peptide and to the description of a protein, and vice versa. The term applies to both naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked by covalent peptide bonds.

[0041] The term "recombinant host cell line" or "host cell" means a cell that contains a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-factor, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate 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.

[0042] The term "operably linked" refers to a functional linkage between two or more elements, and the elements that are operably linked may be adjacent or non-adjacent.

[0043] The term "recombinant plant expression vector" means one or more DNA vectors for achieving plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, together with vectors having a helper plasmid, are most commonly used for Agrobacterium tumefaciens-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be able to express in plant cells, heterologous DNA sequences to be transcribed, etc.

[0044] The term "transformation" refers to a method of introducing a heterologous DNA sequence into a host cell or organism.

[0045] 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

[0046] Figure 1 For OsWRKY15 Detection results of the relative expression levels of the gene after treatment with 20% PEG6000 to simulate drought stress or ABA treatment; wherein, Figure 1 -(a) is OsWRKY15 Detection results of the relative expression levels of the gene after treatment with 20% PEG6000 to simulate drought stress; Figure 1 -(b) is OsWRKY15 Detection results of the relative expression levels of the gene after ABA treatment.

[0047] Figure 2 For OsWRKY15 Identification results of transgenic lines of the rice Figure 2 -(a) is OsWRKY15 Sequence of the gene knockout line; Figure 2 -(b) Sanger sequencing results show that wrky15-1 in lines wrky15-2 and OsWRKY15 in line Figure 2 a single-base deletion and a single-base insertion occurred in the DNA sequence of the gene; OsWRKY15 -(c) is PCR positive identification of the overexpression line of the rice

[0048] Figure 3 For OsWRKY15 Phenotype diagrams of the knockout line of the gene and the wild-type plant WT under normal and drought stress conditions; wherein, Figure 3 -(a) is OsWRKY15 Phenotype results of the gene knockout line under soil drought stress; Figure 3 -(b) is OsWRKY15 Phenotype results of the gene knockout line under 20% PEG6000 stress; Figure 3 -(c) is OsWRKY15 Survival rate of the gene knockout line under soil drought; Figure 3 -(d) is OsWRKY15 Survival rate of the gene knockout line under 20% PEG6000 stress.

[0049] Figure 4 For OsWRKY15 Phenotype diagrams of the overexpression line of the gene and the wild-type plant WT under normal and drought stress conditions; wherein Figure 4-(a) is OsWRKY15 Phenotypic results of gene overexpression lines under soil drought stress; Figure 4 -(b) is OsWRKY15 Survival rate of gene overexpression lines under soil drought stress (OE1); Figure 4 -(c) is OsWRKY15 Survival rate of gene overexpression lines under soil drought stress (OE2). Detailed implementation manners

[0050] 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 progresses. However, it should be understood that the described embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0051] Experimental example 1 OsWRKY15 Test on the expression level of the gene after treatment with 20% PEG6000 simulated drought stress or ABA

[0052] 1 Test method

[0053] 1.1 Sample collection and treatment

[0054] Select plump and uniform Zhonghua 11 rice seeds as wild-type material seeds, place them in an oven at 50 °C for two days to break dormancy, and then surface sterilize them in 0.5% sodium hypochlorite solution for 20 minutes. Rinse them with tap water three times again, soak them in water and then place them in an artificial climate chamber at 28 °C for germination for about 3 days. When the seeds germinate 2-3 mm, transfer them to a cut-bottom PCR plate and float them on tap water (pH value 5.0-5.5) for growth. After one week, transfer them to Yoshida culture medium (Coolaber, NSP1040) for cultivation, and adjust the pH value to 5.0-5.5. The culture medium is changed once a week. When the seedlings grow to the three-leaf stage, apply 20% PEG6000 or ABA to the culture medium respectively, and take samples of the above-ground parts after simulating drought stress treatment for 0 h, 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h and 48 h. Take 5 plants each time. After the materials are frozen in liquid nitrogen, store them in a -80 °C refrigerator for standby.

[0055] 1.2 Extraction of total RNA from rice and RT-PCR detection

[0056] 1.2.1 Extraction of total RNA from plant tissues

[0057] (1) Grind the plant tissue samples frozen with liquid nitrogen into powder, aliquot about 150 mg per tube into RNase-free tubes, add 1 mL of TRIzol to each tube of experimental samples, shake well for 5 min, and let stand on ice for 5 min.

[0058] (2) Add 200 μL of chloroform to each tube for extraction, shake well for 2 min, and let stand on ice for 2 min.

[0059] (3) Centrifuge at 12,000 g at 4 °C for 10 min, and pipette 400 μL of the supernatant into a new RNase-free tube.

[0060] (4) Add 400 μL of isopropanol to each tube, mix well, and place in a -20 °C refrigerator for 2 h.

[0061] (5) Centrifuge at 12,000 g at 4 °C for 10 min, discard the supernatant, add 1 mL of pre-cooled 75% ethanol to suspend the precipitate; centrifuge at 12,000 g at 4 °C for 3 min, discard the supernatant, and repeat twice.

[0062] (6) Place the centrifuge tube on ice and dry it in a laminar flow hood for 10 min.

[0063] (7) Dissolve the precipitate in 30 μL of DEPC water per tube, which can be used for RNA reverse transcription or stored at -80 °C for later use.

[0064] 1.2.2 Reverse transcription of RNA into cDNA

[0065] (1) Measure the RNA concentration with Nanodrop, and add 1 μg of RNA to the reverse transcription system according to the concentration.

[0066] (2) Add the reverse transcription reagent volume (ABclonal, China) to each system: 4 μL of 5×ABScript III RTMix, 1 μL of 20×gDNA Remover Mix, and make up to 20 μL with Nuclease-free H2O.

[0067] (3) Perform reverse transcription reaction on a PCR instrument according to the following reaction program: 37 °C for 2 min, 55 °C for 15 min, 85 °C for 5 min, and hold at 4 °C.

[0068] (4) Measure the cDNA concentration with Nanodrop and store at -20 °C for later use.

[0069] 1.2.3 Real-time fluorescence quantitative PCR (RT-qPCR)

[0070] The nucleotide sequences of the primers used in real-time fluorescence quantification are as follows:

[0071] OsWRKY15 -RT-F: GAGCAAGCAGGTGAAGGTGA (SEQ ID NO.4);

[0072] OsWRKY15 -RT-R: TGGTACACGACGTCGAAGAG (SEQ ID NO.5).

[0073] OsUBQ -RT-F: GCTCCGTGGCGGTATCAT (SEQ ID NO.6);

[0074] OsUBQ -RT-R: CGGCAGTTGACAGCCCTAG (SEQ ID NO.7).

[0075] Perform RT-PCR reactions using a real-time fluorescence PCR instrument according to the following reaction system (Table 1) and running program (Table 2); calculate the relative gene expression levels using the ΔΔCt method (Livak, K.J., and Schmittgen, T.D. (2001). Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 −ΔΔCT Method.Methods. 25, 402–408). OsWRKY15 The gene relative to OsUBQ gene expression levels.

[0076] Table 1 Reaction system for RT-qPCR

[0077]

[0078] Table 2 Running program for RT-qPCR

[0079]

[0080] 2 Experimental results

[0081] OsWRKY15 The changes in gene expression levels after 20% PEG6000 simulated drought stress treatment or ABA treatment are as Figure 1 shown. By detecting the changes in the expression levels of the OsWRKY15 gene over time, it was found that after 20% PEG6000 treatment ( Figure 1 -(a)) or ABA treatment ( Figure 1 -(b)), OsWRKY15The relative expression level of the gene increased, that is OsWRKY15 The gene was up-regulated by drought induction, indicating OsWRKY15 The gene is involved in the response of rice to drought stress.

[0082] Experimental Example 2 OsWRKY15 Genetic transformation and identification test of the gene in rice

[0083] 1 OsWRKY15 Construction of the CRISPR knockout vector for the gene

[0084] According to OsWRKY15 the cDNA sequence of the gene, the target site for knockout was designed using the website: http: / / skl.scau.edu.cn / dsdecode / .

[0085] The nucleotide sequence of the sgRNA target site is: ACCGGTCTATAAGCATCGCGAGG (SEQ ID NO.8).

[0086] The experimental method and vector refer to 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 Dicot Plants. Molecular Plant, 2015, 8(8):1274 - 1284). The recombinant vector was named pYLCRISPR / Cas9Pubi-OsWRKY15.

[0087] OsWRKY15The nucleotide sequence of the CDS of the gene is shown in SEQ ID NO.1: ATGGCCGCCGGAGCGGACGAGCGGTGCCGCGCGCTGGTGTCCGGCCTGCTGTCGTCGATCGACCGGTCTATAAGCATCGCGAGGTCGTGCTGCACGGAGGCCGCCGCCGCCGGGCGCCTGACCCAGCAGGCCGGCGCCGCGCCTGAGTCGCCGCCGTCCGCCGACGGTAGCGCCGGCTCGGACCTCGGCGCCGACTCCCGGTGCCGCGCCAATGCGGCGGGGCCGTGCAAGAAGAGGAAGACGCTGCCCAAGTGGAGCAAGCAGGTGAAGGTGAGGTCCGTGCAGGACGTCGGCCCCCTCGACGACGGGTTCAGCTGGAGGAAGTACGGGCAGAAGGACATCCTCGGCGCCAAGTACCCAAGAGCCTACTTCCGGTGCACGCACCGGCACACGCAGGGCTGCCACGCCAGCAAGCAGGTGCAGCGCGCGGACGGCGACCCGCTGCTCTTCGACGTCGTGTACCACGGCGACCACACCTGCGCGCATGGCGTGCGCTCCGCCGCCGCCGCCATCGACGGCCAGGCCGCGGCGAGCGCGGAGCAAAAGCATCAGCCGACGCCGCCGCAGGAGCAGAACGCCGTCTCGGTCGCGTTCACCTCCATGGCCGTCGTCAACGCATCGACGTCGTCGCCCTTCGTGTCCCCGGCGATGTCGGACTGCCAGATTAGCTACGAGCTGGGCGGTGGCAGCATGGCCGGAGTCCGCAACGTGCCCGACGTGGAGCTTGCCTCCAAGACGAACTCCTCCATGGGAGACGACATGGAGTTCATGTTCTCGCTGGACTCCGATTTCTTGGACACCTACAAGTACTCCAGCTATTTCTAA (SEQ ID NO.1).

[0088] The amino acid sequence of the OsWRKY15 protein is shown in SEQ ID NO.2: MAAGADERCRALVSGLLSSIDRSISIARSCCTEAAAAGRLTQQAGAAPESPPSADGSAGSDLGADSRCRANAAGPCKKRKTLPKWSKQVKVRSVQDVGPLDDGFSWRKYGQKDILGAKYPRAYFRCTHRHTQGCHASKQVQRADGDPLLFDVVYHGDHTCAHGVRSAAAAIDGQAAASAEQKHQPTPPQEQNAVSVAFTSMAVVNASTSSPFVSPAMSDCQISYELGGGSMAGVRNVPDVELASKTNSSMGDDMEFMFSLDSDFLDTYKYSSYF (SEQ ID NO.2).

[0089] OsWRKY15

[0090] 2 OsWRKY15 Construction of gene overexpression vector

[0091] The overexpression vector was constructed by the company, and the recombinant vector was named pBWA(V)HS-OsWRKY15.

[0092] 3 Agrobacterium transformation

[0093] The knockout vector pYLCRISPR / Cas9Pubi-OsWRKY15 and the overexpression vector pBWA(V)HS-OsWRKY15 were respectively transferred into competent cells of Agrobacterium tumefaciens EHA105 by the 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 transgenicexpression of OsiICK6 on plant growth and development, 2011, Annals ofBotany, 107:1087-1101), and the method was referred to the Molecular Cloning Experiment Guide.

[0094] 4 Genetic transformation

[0095] Using the Agrobacterium-mediated genetic transformation method, Zhonghua 11 in rice was used as the receptor material for genetic transformation, and the medium formula is shown in Table 3.

[0096] Table 3 Media and their formulas used for genetic transformation

[0097]

[0098] The specific method is as follows:

[0099] (I) Callus induction

[0100] Take an appropriate amount of mature rice seeds. After dehulling, first wash and disinfect them with 70% alcohol for 1 min, shaking constantly during this period, and then disinfect them with 15% sodium hypochlorite for 30 min (can be placed on a shaker for oscillation); finally, rinse them 4 - 5 times with sterile distilled water, and inoculate them after blotting the surface moisture of the seeds with sterile filter paper. Inoculate the disinfected seeds into an induction medium containing 2.0 mg / L of 2,4-D, and culture them in the dark at 28°C for 30 - 40 days. Expand the callus obtained from the culture on the subculture medium, and subculture it once every 2 weeks until the formation of embryogenic callus.

[0101] (II) Agrobacterium tumefaciens infection

[0102] (1) Streak the Agrobacterium tumefaciens carrying the knockout vector pYLCRISPR / Cas9Pubi-OsWRKY15 and the overexpression vector pBWA(V)HS-OsWRKY15 on the surface of LB solid medium containing antibiotics (50 mg / L kanamycin, 25 mg / L rifampicin), and culture it overnight at 28°C with 200 rpm.

[0103] (2) Pick monoclonal colonies with a sterilized toothpick and inoculate them into 5 mL of LB liquid medium (Coolaber, PM0010) containing antibiotics (50 mg / L kanamycin, 25 mg / L rifampicin), and culture them with shaking at 28°C until OD 600 = 0.5.

[0104] (3) Inoculate the activated fresh bacterial liquid into 25 mL of the same LB liquid medium at a ratio of 1:100, and culture it under the same conditions until OD 600 = 0.5.

[0105] (4) Centrifuge the bacterial liquid at 5000 g and 4°C for 10 min to collect the bacteria, discard the supernatant; add 25 mL of 10 mM MgSO4 to suspend the bacteria, and gently pipette to make it fully suspended, then centrifuge at 5000 g and 4°C for 10 min again to collect the bacteria, and discard the supernatant.

[0106] (5) Resuspend it with 25 mL of infection medium (AA-AS) containing 200 μM acetosyringone (AS).

[0107] (6) Transfer the embryogenic callus with good growth state from the subculture medium to a petri dish covered with sterile filter paper (cut the callus into pieces of 0.3 - 0.4 mm in size), and air-dry it on the ultra-clean workbench for 10 - 20 min.

[0108] (7) Immerse the dried embryogenic callus into a 50 mL centrifuge tube containing the above-mentioned bacterial solution for 20 min, shaking it every 5 min during this period; pour out the bacterial solution, take out the callus and place it on a sterile filter paper to air dry for 10 - 20 min, and then transfer it to a co-culture medium (CC) containing 200 μM acetosyringone (AS) with a sterile filter paper on the surface, and co-culture it for 3 days under the condition of 25℃ in the dark.

[0109] (8) Collect the callus without obvious Agrobacterium on the surface, rinse it 3 times with sterile water containing 600 mg / L cefotaxime, and suck out the excess moisture.

[0110] (9) Transfer the callus to a selection medium (N6 medium containing 500 mg / L cefotaxime and 50 mg / L hygromycin) for continuous screening 2 - 3 times, each time for two weeks. Finally, obtain bright yellow hygromycin-resistant callus with good growth.

[0111] (III) Regeneration of transgenic plants

[0112] Take fresh hygromycin-resistant callus, divide the callus into small pieces of 2 mm, inoculate them into the pre-regeneration medium, and culture them in the dark at 28℃ for 7 days, then place them in a light culture room (12 h light / 12 h dark) and continue to culture for 8 - 9 days. Then transfer the callus with adventitious buds differentiated to the regeneration medium (250 mL tissue culture flask) and continue light culture. Wait until the adventitious buds grow into small seedlings 4 - 6 cm high, and then transfer them to the rooting medium and culture them in the light culture room at 28℃ (12 h light / 12 h dark) for about 15 days to obtain transgenic plants, transfer them to the greenhouse for planting (T0 generation), take leaves one month later and perform PCR positive identification using primers Hyg-F / R, and harvest the seeds of the positive transgenic plants (T1 generation) of the identified positive transgenic plants.

[0113] The nucleotide sequences of primers Hyg-F / R are shown as follows:

[0114] Hyg-F: CAAAGATCGTTATGTTTATCGGCACT (SEQ ID NO.9);

[0115] Hyg-R: TTGGCGACCTCGTATTGGGAA (SEQ ID NO.10).

[0116] 5 TPS method for extracting rice genomic DNA

[0117] (1) Take plant leaves and put them into a 2 mL centrifuge tube, add steel beads with a diameter of 5 mm to each tube, quickly freeze them in liquid nitrogen, and grind the samples with a tissue grinder at 1500 rpm for about 90 s;

[0118] (2) Add 1000 μL of TPS extraction solution to each tube, mix well, place in an oven at 65°C for 30 min, and invert and mix twice in the middle;

[0119] (3) Centrifuge at 12,000 g for 10 min at room temperature, and pipette approximately 500 μL of the supernatant into a new 1.5 mL centrifuge tube;

[0120] (4) After gently mixing with 400 μL of isopropanol, let it stand at room temperature for 10 min;

[0121] (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;

[0122] (6) Dry in a laminar flow hood, add 50 μL of ddH2O to dissolve the DNA, and store at 4°C for later use.

[0123] 6 OsWRKY15 Identification of Gene-Edited Rice and Analysis of sgRNA Target Editing

[0124] Extract the genomic DNA of T2 generation OsWRKY15 gene-edited lines, and design primers OsWRKY15-KO-F / R centered on the target site to amplify the sequence. The PCR amplification products were sequenced. Through sequence alignment analysis of the sequencing results, two different gene-edited materials were obtained. Compared with the nucleotide sequence at the target site of the wild type (SEQ ID NO.8), wrky15-1 the line deleted 1 base at the target site (ACCGGTCTATAAGCATGCGAGG (SEQ ID NO.13)); wrky15-2 the line inserted 1 base at the target site (ACCGGTCTATAAGCATCAGCGAGG (SEQ ID NO.14)). The deletion or insertion of bases in the above two lines will cause frameshift mutations, resulting in protein sequences without conserved domains, making the OsWRKY15 protein functionless. The two OsWRKY15- KO lines can be used for subsequent phenotype observation experiments ( Figure 2 -(a), Figure 2 -(b)).

[0125] The nucleotide sequences of primers OsWRKY15-KO-F / R are as follows:

[0126] OsWRKY15-KO-F: GAGCGGACGAGCGGTG (SEQ ID NO.11);

[0127] OsWRKY15-KO-R: TAGAAACCGGTCAACGTGCC (SEQ ID NO.12).

[0128] 7 OsWRKY15 Identification of gene overexpression lines

[0129] Extract the genomic DNA of the T2 generation OsWRKY15 gene overexpression lines, and amplify the sequence with primers OsWRKY15-OE-F / R. Run the PCR amplification products on a gel, and the lines with bands are overexpression positive lines. Select two OsWRKY15 - OE lines that can be used for subsequent phenotypic observation experiments ( Figure 2 -(c)).

[0130] OsWRKY15-OE-F: AGTGGAAAAGGAAGGTGGCT (SEQ ID NO.15);

[0131] OsWRKY15-OE-R: CGTCTTCCTCTTCTTGCACG (SEQ ID NO.16).

[0132] Experimental Example 3 OsWRKY15 Phenotypic identification test of gene overexpression and knockout lines after drought treatment

[0133] 1 Phenotypic identification test of 20% PEG6000 simulated drought stress treatment

[0134] To clarify OsWRKY15 the tolerance of gene knockout materials to drought stress, select plump and uniform wild-type and knockout mutant material seeds, and place them in an oven at 50 °C for two days to break dormancy. Then surface sterilize them in 0.5% sodium hypochlorite solution for 20 minutes. Rinse them with tap water three times again, soak them in water and then place them in an artificial climate chamber at 28 °C for germination for about 3 days. When the seeds germinate 2-3 mm, transfer them to a bottom-cut PCR plate and float them on tap water (pH value 5.0-5.5) to grow. After one week, transfer them to Yoshida culture solution and adjust the pH value to 5.0-5.5. Replace the culture solution every Monday. Apply 20% PEG6000 to the culture solution of seedlings grown to the three-leaf stage. Set three replicates for the treatment, with 24 seeds of wild-type and knockout materials in each replicate. Observe the phenotype 14 days after drought stress treatment.

[0135] The test results are as Figure 3 shown. Compared with wild-type rice WT, OsWRKY15 the gene knockout mutant plants are more sensitive to drought stress ( Figure 3 -(b)), OsWRKY15The survival rate of the gene knockout plants is also lower than that of the wild type WT ( Figure 3 -(d)), indicating that knocking out the rice OsWRKY15 gene can significantly affect the drought stress resistance ability of rice.

[0136] 2 Soil drought phenotype identification experiment

[0137] To confirm OsWRKY15 that the gene positively regulates rice drought resistance, the seeds of the 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 flower pots containing a mixture of nutrient soil and vermiculite. The flower pots were placed in a container filled with water and grown in the sun for 1 month (three-leaf stage) in summer (25 - 35 °C). Under the condition of not watering, drought stress was applied until the leaves and stems withered (about 7 d), and then watering was resumed for 7 d. The survival rates of each line were counted. The transgenic lines and the control lines were planted in the same pot.

[0138] OsWRKY15 The experimental results of the knockout lines of the Figure 3 gene are as OsWRKY15 shown. The wilting and withering degrees of the gene knockout plants are greater than those of the wild type WT ( Figure 3 -(a)), OsWRKY15 and the survival rate of the gene knockout plants is also lower than that of the wild type WT ( Figure 3 -(c)). Therefore, after knocking out the mutant rice OsWRKY15 gene in rice, the plants are more sensitive to drought stress.

[0139] OsWRKY15 The experimental results of the overexpression lines of the Figure 4 gene are as OsWRKY15 shown. The wilting and withering degrees of the gene overexpression plants are less than those of the wild type WT ( Figure 4 -(a)), OsWRKY15 and the survival rate of the gene overexpression plants is also higher than that of the wild type WT ( Figure 4 -(b), Figure 4 -(c)). Therefore, after overexpressing the rice OsWRKY15 gene in rice, the plants are more drought-resistant. It shows that OsWRKY15 the gene positively regulates rice drought resistance. Therefore, the rice OsWRKY15 gene and its encoded protein have the ability to enhance the drought stress resistance of crops.

Claims

1. OsWRKY15 Gene, OsWRKY15 protein, containing OsWRKY15 Gene expression cassette or containing OsWRKY15 Use of a recombinant plant expression vector of a gene in improving plant resistance to drought stress; the plant is rice; 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.

2. The use according to claim 1, characterized in that The improving the resistance of plants 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.

3. The use according to claim 1 or 2, 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.

4. 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 plant is rice.

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