Method for improving drought resistance of upland rice by regulating oswrky31 gene

Abstract

The application discloses a method for improving drought resistance of upland rice by regulating an OsWRKY31 gene, and belongs to the technical field of genetic breeding. Oswrky31 The amino acid of the protein coded by the OsWRKY31 gene is SEQ ID NO:1. The mutant provided by the application is obtained by CRISPR-Cas9 technology, and the change of the nucleotide of the mutant causes early termination of protein translation. Oswrky31 The mutant has higher survival rate and less active oxygen accumulation, and the drought resistance of the rice and the upland rice is improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic breeding technology, specifically involving a method for improving the drought resistance of upland rice by regulating the OsWRKY31 gene. Background Technology

[0002] As a core global food crop, rice's stable and increased yields are crucial for ensuring food security. Compared to conventional rice, which relies on paddy field irrigation, upland rice has lower water requirements and is more suitable for two key scenarios: regions with insufficient freshwater resources and rain-fed agriculture areas lacking adequate irrigation facilities. The application scope of upland rice is constantly expanding, and its planting area is gradually increasing. In recent years, climate change has led to frequent extreme drought events, seriously threatening the production stability of both rice and upland rice. Drought stress not only affects photosynthesis and nutrient absorption in both crops but also interferes with reproductive development, leading to significant yield reductions. Traditional drought-resistant breeding relies on the aggregation of quantitative trait loci, but this is often accompanied by trait linkage defects and insufficient environmental adaptability. Breakthroughs at the molecular mechanism level are urgently needed to cultivate new varieties that combine drought resistance with high-yield potential.

[0003] WRKY transcription factors are one of the largest families of transcriptional regulators in plants, playing crucial roles in plant growth, development, senescence, and both abiotic and biotic stresses. At the DNA level, WRKY transcription factors can bind to the W-box TTGAC (C / T) in the promoters of target genes, regulating downstream gene expression through self-regulation or cross-regulation. At the protein level, WRKY transcription factors can interact with various proteins, including MAP kinases, histone deacetylases, resistance R proteins, and various transcription factors, regulating plant growth, development, and various stress responses. Overexpression of OsWRKY31 enhances disease resistance in rice but affects root growth and auxin response. Overexpression of OsWRKY31 reduces sensitivity to high concentrations of IBA, NAA, and 2,4-D. OsWRKY31 may be a common component in auxin response signaling pathways and defense response signaling pathways in both rice and upland rice; therefore, in-depth research into the function and regulatory mechanisms of this gene will have significant theoretical and practical value. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide new uses for OsWRKY31. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: This invention provides the use of a protein, or a substance that inhibits, reduces, or downregulates the expression of a gene encoding said protein, or a substance that inhibits, reduces, or downregulates the activity or content of said protein, in any of the following: C1) Improve the drought resistance of plants; C2) Prepare products that improve plant drought resistance; C3) Cultivate plants with improved drought resistance; C4) Prepare products from plants with improved drought resistance; C5) Plant breeding or assisted plant breeding.

[0006] The protein is OsWRKY31, and OsWRKY31 is any of the following: A1) The amino acid sequence is that of the protein SEQ ID NO:1; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

[0007] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0008] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0009] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0010] In the above applications, the substance is any one of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the aforementioned protein, or RNA molecules that knock out the gene encoding the aforementioned protein. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7) Transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) A transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

[0011] The present invention also provides any of the following applications of biomaterials related to the aforementioned proteins: C1) Improve the drought resistance of plants; C2) Prepare products that improve plant drought resistance; C3) Cultivate plants with improved drought resistance; C4) Prepare products from plants with improved drought resistance; C5) Plant breeding or assisted plant breeding; The biomaterial is any one of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the aforementioned protein, or RNA molecules that knock out the gene encoding the aforementioned protein. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7) Transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) A transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

[0012] The present invention also provides a method for improving the drought resistance of plants by inhibiting, reducing or downregulating the expression of the gene encoding the protein in the recipient plant or inhibiting, reducing or downregulating the activity and / or content of the protein, thereby improving the drought resistance of the recipient plant, wherein the recipient plant contains the gene encoding the protein. In the above-mentioned methods for improving the drought resistance of plants, the inhibition, reduction, or downregulation of the expression of the coding gene of the aforementioned protein in the recipient plant, or the inhibition, reduction, or downregulation of the activity and / or content of the protein, includes introducing into the recipient rice a substance that inhibits, reduces, or downregulates the expression of the coding gene of the aforementioned protein in the recipient plant, or inhibits, reduces, or downregulates the activity and / or content of the protein, or knocks out the coding gene of the aforementioned protein in the recipient plant.

[0013] In the above methods for improving the drought resistance of plants, the substance is any one of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the aforementioned protein, or RNA molecules that knock out the gene encoding the aforementioned protein. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4).

[0014] In some specific embodiments of the present invention, the gene encoding the protein in the knockout recipient plant includes introducing a gene knockout vector into the recipient plant with the reverse complementary sequence of nucleotides 730-749 of SEQ ID NO:3 and / or nucleotides 846-865 of SEQ ID NO:3 as the target site.

[0015] The present invention also provides a method for cultivating plants with improved drought resistance, by inhibiting, reducing or downregulating the expression of the coding genes of the aforementioned proteins in the starting plant or regulating the activity and / or content of the proteins to obtain a target plant, wherein the target plant has higher drought resistance than the starting plant.

[0016] In the above-mentioned method for cultivating plants with improved drought resistance, the inhibition, reduction, or downregulation of the expression of the coding gene of the aforementioned protein in the starting plant, or the inhibition, reduction, or downregulation of the protein activity and / or content, includes introducing into the recipient rice a substance that inhibits, reduces, or downregulates the expression of the coding gene of the aforementioned protein in the starting plant, or inhibits, reduces, or downregulates the protein activity and / or content, or knocks out the coding gene of the aforementioned protein in the starting plant.

[0017] In the above-mentioned method for cultivating plants with improved drought resistance, the substance is any one of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the aforementioned protein, or RNA molecules that knock out the gene encoding the aforementioned protein. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4).

[0018] In some specific embodiments of the present invention, knocking out the gene encoding the protein in the starting plant includes introducing a gene knockout vector into the starting plant with the reverse complementary sequence of nucleotides 730-749 of SEQ ID NO:3 and / or nucleotides 846-865 of SEQ ID NO:3 as the target site.

[0019] In the above method, knocking out the coding gene in the target plant involves mutating the coding gene in the target plant as follows: K1) Delete nucleotides 734 to 738 of SEQ ID NO:3; K2) Delete nucleotides 852 to 884 of SEQ ID NO:3; K3) Delete nucleotides 734 to 750 of SEQ ID NO:3; K4) An A is inserted between nucleotides 859 and 860 of SEQ ID NO:3; K5) Delete nucleotides from position 733 to 860 of SEQ ID NO:3; K6) An A is inserted between nucleotides 734 and 735 of SEQ ID NO:3; K7) Delete nucleotides 858 to 859 of SEQ ID NO:3; This invention provides a biomaterial, wherein the biomaterial is at least one of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the aforementioned protein, or RNA molecules that knock out the gene encoding the aforementioned protein. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7) Transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) Transgenic plant organs containing the gene described in B2), or transgenic plant organs containing the expression cassette described in B3), or transgenic plant organs containing the recombinant vector described in B4); B9) A transgenic plant containing the gene described in B2), or a transgenic plant containing the expression cassette described in B3), or a transgenic plant containing the recombinant vector described in B4).

[0020] In some specific embodiments of the present invention, the transgenic plant described in B9) includes any of the following: P1) Compared with wild-type rice Nipponbare, the mutant plants Oswrky31-1Nucleotides 734 to 738 (ACGAA) of SEQ ID NO:3 were deleted, and nucleotides 852 to 884 (5'-AGACTGCATCTGGGAAGGCAGTCTCCATCTGCT-3') of SEQ ID NO:3 were deleted. These nucleotide changes caused premature termination of protein translation.

[0021] P2) Compared with wild-type rice Nipponbare, the mutant plants Oswrky31-2 Nucleotides 734 to 750 (5'-ACGAAGCAGATTCAGCA-3') of SEQ ID NO:3 were deleted, and an A was inserted between nucleotides 859 and 860 of SEQ ID NO:3. These nucleotide changes caused premature termination of protein translation.

[0022] Compared to wild-type dryland rice (P3), Oswrky31-3 Nucleotides 733 to 860 (5'-AACGAAGCAGATTCAGCAGAAAGATTATAGTGATCCACCATTGTACTCAGTCACCTACTACAATGAGCATACATGTAATAGTGCTTTTCTTCCTCTTAGCCCCTCAGAGTTCCAGCTGCAGACTGCAT-3') of SEQ ID NO:3 were deleted, and the above nucleotide changes caused premature termination of protein translation.

[0023] P4) Compared with wild-type dryland rice Oswrky31-4 The nucleotides 734 to 735 of SEQ ID NO:3 have an insertion of an 'A' between them, and the nucleotides 858 to 859 of SEQ ID NO:3 have a deletion (CA). These changes in nucleotides cause premature termination of protein translation.

[0024] In this article, the plant referred to is any of the following: G1) Monocotyledonous or dicotyledonous plants; G2) Plants of the order Poales; G3) Gramineae plants; G4) Plants of the genus *Oryza*; G5) Rice plants.

[0025] In some specific embodiments of the present invention, the rice plant is paddy rice.

[0026] In some specific embodiments of the present invention, the rice plant is upland rice.

[0027] The expression cassette described herein refers to a DNA molecule capable of expressing the proteins described above in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all the regulatory sequences necessary for the expression of any of the aforementioned proteins. The regulatory sequences, under compatible conditions, guide the coding sequence to express any of the aforementioned proteins in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of a protein that guides the encoded protein into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein into the secretory pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory systems are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that can amplify genes. In these examples, the nucleic acid sequence encoding the protein should be operatively linked to the regulatory sequence.

[0028] In this article, the vector may be a plasmid, granule, bacteriophage, or viral vector.

[0029] In some specific embodiments of the present invention, the carrier is specifically pYLCRISPR / Cas9Pubi-H.

[0030] In this article, the microorganisms mentioned may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0031] In this article, the transgenic plant cell lines mentioned do not include propagation material.

[0032] In some specific embodiments, the dryland rice is specifically dryland rice IRAT109.

[0033] "Transgenic plant" (transgenic upland rice) refers to a plant whose genome has been altered by integrating or inserting recombinant DNA molecules, constructs, cassettes, or sequences for the expression of non-coding RNA molecules, mRNA, and / or proteins. Transgenic plants include R0 generation plants developed or regenerated from initially transformed plant cells and their offspring, or plants obtained by crossing with R0 generation transgenic plants containing recombinant DNA molecules, constructs, cassettes, or sequences. Plants with integrated or inserted recombinant DNA molecules, constructs, cassettes, or sequences are considered transgenic plants, even if the plant also has other mutations or edits that are not themselves considered transgenic.

[0034] "Plant cells" (rice cells) are biological cells of plants, which are taken from plants or derived from cultures obtained by culturing cells taken from plants.

[0035] "Transgenic plant cells" (transgenic upland rice cells) refer to any plant cell transformed with a stably integrated recombinant DNA molecule, construct, cassette, or sequence. Transgenic plant cells can include original transformed plant cells, transgenic plant cells regenerated or developed from R0 generation transgenic plant cells, transgenic plant cells cultured from another transgenic plant cell, or transgenic plant cells from any progeny or offspring of a transformed R0 generation plant, including cells of plant seeds or embryos, or cultured plant cells, callus cells, etc.

[0036] The plants described in this application can be viable, non-viable, renewable, and / or non-renewable. The plants described in this application include propagules or propagation material. "Propagules or propagation material" can include any plant part that can grow into a whole plant. "Plant part" can refer to any organ or tissue of a plant, such as meristematic tissue, bud organs / structures (e.g., leaves, stems, or nodes), roots, flowers or floral organs / structures (e.g., flowers, bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (e.g., embryo, endosperm, and seed coat), fruits (e.g., mature ovaries), propagules or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, etc.) or any part thereof.

[0037] In some embodiments, the OsWRKY31 The coding region sequence of the gene is SEQ ID NO:2.

[0038] In some embodiments, the CRISPR-Cas9 system uses the OsWRKY31 The primer sequences for the gRNA target sites after gene adapter addition are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

[0039] In some embodiments, the method obtains knockout mutants in both rice and upland rice backgrounds by introducing the coding gene for the sgRNA targeting the target sequence and the coding gene for Cas9 into rice. Oswrky31 .

[0040] In some embodiments, the CRISPR / Cas9 vector contains a hygromycin resistance gene, facilitating the screening of positive plants after transformation. The Agrobacterium strain EHA105, known for its high transformation efficiency, was selected, and the constructed gene-editing vector was electroporated into Agrobacterium cells. Well-prepared callus tissue was collected, and an appropriate amount of the bacterial culture to be transformed was added until the callus tissue was completely submerged, promoting efficient T-DNA transfer. After co-culture, the transgenic-induced callus tissue was transferred to a solid selection medium containing hygromycin to inhibit Agrobacterium growth and screen for transformed cells. Through steps such as induction and differentiation culture of well-prepared callus tissue, complete transgenic regenerated plants were obtained. The resulting seedlings were then transferred to a rooting medium for rooting and seedling strengthening, and finally transplanted to an experimental field for growth. Hygromycin and Cas9 gene-specific primers were used to detect the DNA of the transgenic regenerated plants, and transgenic positive materials were screened. Subsequent next-generation sequencing analysis was performed to identify whether gene editing had occurred at the target site, as well as the type and efficiency of the editing.

[0041] In some embodiments, the knockout mutants in rice and upland rice backgrounds Oswrky31 Features include increased survival rate and reactive oxygen species scavenging capacity under drought stress, as well as enhanced drought resistance.

[0042] This invention utilizes CRISPR-Cas9-mediated precision editing technology to edit WRKY family genes in rice and upland rice without introducing exogenous DNA. OsWRKY31 They created a knockout mutant. Oswrky31 This study aims to enhance the drought adaptability of rice and upland rice, provide new molecular targets for crop drought resistance genetic improvement, and is of great significance for ensuring the sustainable development of agriculture in arid regions. Attached Figure Description

[0043] Figure 1 Rice background mutant plants Oswrky31-1 and Oswrky31-2 The mutation type. Starting from the first base of the start codon, in the mutant plant... Oswrky31-1middle OsWRKY31 The CDS coding sequence in the gene contains a 5-base deletion at position 205 and a 33-base deletion at position 323. In the mutant plant... Oswrky31-2 middle OsWRKY31 The CDS coding sequence in the gene has a deletion of 17 bases at position 205 and an insertion of 1 base at position 331. These nucleotide changes cause premature termination of protein translation, resulting in a knockout mutant.

[0044] Figure 2 Mutant plants in the background of drought-resistant rice Oswrky31-3 and Oswrky31-4 The mutation type. Starting from the first base of the start codon, in the mutant plant... Oswrky31-3 middle OsWRKY31 A 128-base deletion exists at position 204 of the CDS coding sequence in the gene. In mutant plants... Oswrky31-4 middle OsWRKY31 An insertion of one base at position 206 and a deletion of two bases at position 329 in the CDS coding sequence of the gene cause premature termination of protein translation, resulting in a knockout mutant.

[0045] Figure 3 Rice wild-type Nipponbare and its mutant Oswrky31 The drought resistance phenotype, survival rate, and leaf water content of rice were observed. Wild-type rice seedlings and mutant seedlings at the four-leaf stage were drought treated for 15 days, followed by a recovery treatment for 5 days. The drought resistance phenotype of the two types was observed (A), and the difference in seedling survival rate (B) and leaf water content on the day before the phenotype was photographed were recorded (C).

[0046] Figure 4 Wild-type IRAT109 and mutant rice Oswrky31 The drought resistance phenotype, survival rate, and leaf water content of rice were observed. Wild-type rice seedlings and mutant seedlings at the four-leaf stage were drought treated for 18 days, followed by a recovery treatment for 5 days. The drought resistance phenotype of the two types was observed (A), and the difference in seedling survival rate (B) and leaf water content on the day before the phenotype was photographed were recorded (C).

[0047] Figure 5 A wild-type Nipponbare rice variety at the four-leaf stage and its mutant Oswrky31 DAB staining before and after drought treatment.

[0048] Figure 6 Wild-type IRAT109 and mutant rice at the four-leaf stage Oswrky31 DAB staining before and after drought treatment. Detailed Implementation

[0049] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0051] The pYLCRISPR / Cas9Pubi-H plasmid used in the following examples was kindly provided by South China Agricultural University. This plasmid is disclosed in "Ma, X., Zhang, Q., Zhu, Q. et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants". Mol Plant 8:1274–1284 (2015). doi: 10.1016 / j.molp.2015.04.007”, the biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of the present invention and may not be used for any other purpose.

[0052] The rice variety Nipponbare in the following examples belongs to the conventional germplasm of temperate japonica rice. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0053] The upland rice IRAT109 in the following examples belongs to the tropical upland japonica rice germplasm, also known as Suyin Rice No. 2, with the approval number Suzhongshenzi No. 158. It belongs to the tropical upland japonica rice germplasm and is disclosed in the article "Xia Zhihong. High-quality japonica upland rice IRAT109 and its cultivation technology [J]. Agricultural and Forestry Science Experiment, 1994(06):5. DOI:CNKI:SUN:ANHE.0.1994-06-000." The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of this invention and cannot be used for other purposes.

[0054] The following examples use GraphPad Prism 9 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. A two-tailed Student's t-test was used, and P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and ns (not significant) were used.

[0055] Example 1: Construction of the mutant knockout vector pYLCRISPR / Cas9Pubi-H-OsWRKY31 OsWRKY31 The genome sequence of the gene is shown in SEQ ID NO:3. In SEQ ID NO:3, positions 120-172 are exon 1, positions 291-395 are exon 2, and positions 688-1162 are exon 3. WRKY31 The CDS coding sequence of the gene is shown in SEQ ID NO:2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:1.

[0056] This invention provides a method for constructing pYLCRISPR / Cas9Pubi-H-OsWRKY31, with the following specific steps: 1. Design and synthesis of gRNA target primers according to OsWRKY31 Based on the gene exon sequences, two pairs of gRNA target primers were designed and synthesized: OsWRKY31-gRNA-OsU3T1 (SEQ ID NO:4), OsWRKY31-gRNA-gRT1 (SEQ ID NO:5), OsWRKY31-gRNA-OsU6aT2 (SEQ ID NO:6), and OsWRKY31-gRNA-gRT2 (SEQ ID NO:7). Detailed information is shown in Table 1. The nucleotide sequence of target 1 is TCCAGCTGCAGACTGCATCT, targeting nucleotides 843-862 of SEQ ID NO:3. The nucleotide sequence of target 2 is AGCAACGAAGCAGATTCAGC, targeting the reverse complementary sequence of nucleotides 730-749 of SEQ ID NO:3. Table 1. Detailed information on the design and synthesis of gRNA target primers

[0057] In OsWRKY31-gRNA-OsU3T1, the lowercase letters correspond to target sequence 1, and the uppercase letters represent the sgRNA backbone sequence; in OsWRKY31-gRNA-gRT1, the lowercase letters correspond to the reverse complementary sequence of target sequence 1, and the uppercase letters represent the sgRNA backbone sequence; in OsWRKY31-gRNA-OsU6aT2, the lowercase letters correspond to the forward sequence of target sequence 2, and the uppercase letters represent the sgRNA backbone sequence; in OsWRKY31-gRNA-gRT2, the lowercase letters correspond to the reverse complementary sequence of target sequence 2, and the uppercase letters represent the sgRNA backbone sequence.

[0058] 2. Construction of sgRNA expression cassette Two target sequences, T1 and T2, were ligated downstream of two Ubiquitin promoters, OsU3 and OsU6a, respectively, to construct two tandem sgRNA expression cassettes. In the two tandem sgRNA expression cassettes, target sequence T1 was located downstream of promoter OsU3, and target sequence T2 was located downstream of promoter OsU6a.

[0059] The nucleotide sequences of YLgRNA-U6 / U3, UF, gR-R, Pps-R, Pgs-2, Pps-2 and Pgs-L are referenced in "Zeng Dongchang et al. 'Operational methods for construction and mutation analysis of plant CRISPR / Cas9 multi-gene editing vectors. Science in China: Life Sciences 48.7 (2018):12'".

[0060] YLgRNA-U3 GenBank accession number: KR029097.

[0061] YLgRNA-U6a GenBank accession number: KR029099.

[0062] UF: 5'-CTCCGTTTTACCTGTGGAATCG-3'; gR-R:5'-CGGAGGAAAATTCCATCCAC-3'; Pgs-2:5'-AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3'; Pps-2:5'-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG-3'; Pgs-L:5'-AGCGTGGGTCTCGCTCGACGCGTATCCATCCACTCCAAGC-3'; This step uses the overlapping PCR method. The first step of PCR introduces the target sequence downstream of the U3 / U6 promoter and upstream of the sgRNA sequence.

[0063] Reaction 1: 7.5 µL of 2× Phanta Max Buffer, 0.25 µL of 10 mmol / L dNTPs Mix, 0.2 U of Phanta Max Polymerase, 2-5 ng of YLgRNA-U3, 0.3 µL each of 10 µmol / L UF and OsWRKY31-gRNA-OsU3T1, and ddH2O to bring the total volume to 15 µL.

[0064] Reaction 2: 7.5 µL of 2× Phanta Max Buffer, 0.25 µL of 10 mmol / L dNTPs Mix, 0.2 U of Phanta Max Polymerase, 2-5 ng of YLgRNA-U3, 0.3 µL each of 10 µmol / L OsWRKY31-gRNA-gRT1 and gR-R, and ddH2O to bring the total volume to 15 µL.

[0065] Reaction 3: 7.5 µL of 2× Phanta Max Buffer, 0.25 µL of 10 mmol / L dNTPs Mix, 0.2 U of Phanta Max Polymerase, 2-5 ng of YLgRNA-U6a, 0.3 µL each of 10 µmol / L UF and OsWRKY31-gRNA-OsU6aT2, and ddH2O to bring the total to 15 µL.

[0066] Reaction 4: 7.5 µL of 2× Phanta Max Buffer, 0.25 µL of 10 mmol / L dNTPs Mix, 0.2 U of Phanta Max Polymerase, 2-5 ng of YLgRNA-U6a, 0.3 µL each of 10 µmol / L OsWRKY31-gRNA-gRT2 and gR-R, and ddH2O to a final volume of 15 µL. Perform 25-26 PCR cycles: 95℃ for 10 s, 58℃ for 15 s, 72℃ for 15 s. Collect 3-5 µL of the PCR product and examine by 1.5% agarose gel electrophoresis (the product from reaction 2 is approximately 140 bp in length).

[0067] In the second step of PCR, the promoter, target site, and sgRNA were constructed into a complete expression cassette. 1 µL each of the products from reactions 1 and 2 of the first round of PCR were added to 8 µL of ddH2O and diluted 10-fold. 1 µL of this product was used as the template for the second round of PCR. The reaction volume was 30 µL. The following were added: 15 µL of 2× Phanta Max Buffer, 0.5 µL of 10 mmol / L dNTP Mix, 0.4 U of PhantaMax, 0.5 µL of 10 µmol / L mixed universal primers (Pps-R / Pgs-2), and 1 µL of the reaction 1 + reaction 2 dilution buffer. ddH2O was then added to bring the volume to 30 µL. PCR cycling was performed at 25-28°C for 10 s, 58°C for 15 s, and 72°C for 20 s. The OsU3-T1 expression cassette was obtained.

[0068] Take 1 µL each of the products from reactions 3 and 4 in the first round of PCR and add them to 8 µL of ddH2O for a 10-fold dilution. Use 1 µL of this product as the template for the second round of PCR. The reaction volume is 30 µL. Add 15 µL of 2× Phanta Max Buffer, 0.5 µL of 10 mmol / L dNTP Mix, 0.4 U of Phanta Max, 0.5 µL of 10 µmol / L mixed universal primers (Pps-2 / Pgs-L), and 1 µL of the reaction 3 + reaction 4 dilution buffer. Make up the total volume to 30 µL with ddH2O. PCR cycling: 95℃ for 10 s, 58℃ for 15 s, 72℃ for 20 s. Obtain the OsU6a-T2 expression cassette.

[0069] 3. The sgRNA expression cassette was cloned into the pYLCRISPR / Cas9Pubi-H vector. Prepare a 15 µL reaction mixture: 1.5 µL of 10× CutSmart Buffer; 1.5 µL of 10 mmol / L ATP (or 1.5 µL of 10× T4 DNAligase buffer can be added instead of ATP), 60-80 ng of pYLCRISPR / Cas9Pubi-H plasmid, 20-30 ng of purified mixed sgRNA expression cassettes per cassette, 10 U of Bsa I-HF, 35 U of T4 DNAligase, and ddH2O to a final volume of 15 µL. Perform 10-15 cycles of ligation-digestion reaction using a variable temperature cycle (PCR instrument can be used) (37℃ for 5 min, 10℃ for 5 min, 20℃ for 5 min); finally, incubate at 37℃ for 5 min to obtain the recombinant vector pYLCRISPR / Cas9Pubi-H-OsWRKY31.

[0070] The recombinant vector pYLCRISPR / Cas9Pubi-H-OsWRKY31 is a recombinant plasmid obtained by tandemly inserting the OsU3-T1 expression cassette and the OsU6a-T2 expression cassette between two BsaⅠ restriction sites of the pYLCRISPR / Cas9 plasmid while keeping the other nucleotide sequences of the pYLCRISPR / Cas9 plasmid unchanged.

[0071] Example 2: Genetic transformation of paddy rice and upland rice, and the acquisition and screening of positive knockout plants. 1. Genetic transformation of rice and upland rice and the acquisition of positive knockout plants All culture medium formulations used in the induction medium, NB solid medium, NB solid screening medium, and rooting medium were based on the reference "Hiei Y and Komari T. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc. 2008:3(5):824–834. https: / / doi.org / 10.1038 / nprot.2008.46".

[0072] (1) Callus was induced and subcultured using mature seeds of Nipponbare rice and IRAT109 upland rice on induction medium. The specific procedures were as follows: Nipponbare rice and IRAT109 upland rice were used as recipients. After removing the husks, the recipient seeds were surface-sterilized with 70% (v / v) ethanol for 1 min, followed by washing with 2.5% (w / v) sodium hypochlorite solution with shaking for 45 min. After rinsing three times with sterile water, the seeds were sown on NB solid medium and cultured at 28°C in the dark for two weeks. Once callus tissue grew from the mature embryo scutellaria, it was excised and subcultured on fresh NB solid medium. The tissue was transferred to fresh NB solid medium every 7 days. After 3-4 transfers, Agrobacterium infection could be performed.

[0073] (2) Culture of Agrobacterium tumefaciens EHA105. When the callus tissue of rice and upland rice was in good condition, the recombinant vector pYLCRISPR / Cas9Pubi-H-OsWRKY31 constructed in Example 1 was transformed into Agrobacterium tumefaciens strain EHA105 by high voltage electroporation. The transformed vector was then spread on LB solid medium containing 50 mg / L kanamycin and 25 mg / L rifampin. After inverted culture for 2-3 days, 4-5 single colonies were picked and inoculated into 5 mL of LB liquid medium containing kanamycin and rifampin. The culture was carried out overnight at 250 rpm at 28°C. The next day, the bacterial cells were centrifuged at 3000 rpm for 5 min at room temperature to precipitate the bacterial cells. The cells were then resuspended in transformation solution for use in the transformation of rice and upland rice callus tissue.

[0074] (3) Agrobacterium EHA105 was used to infect rice and upland rice callus. Healthy callus tissue was collected, and an appropriate amount of the bacterial solution to be transformed was added until the callus tissue was completely submerged. The tissue was left at room temperature for 20 minutes, gently agitated several times during this period. The transformed callus tissue was then removed, and excess bacterial solution was absorbed with sterile filter paper. The tissue was then incubated at 23ºC in the dark for 2-3 days. Subsequently, the transgenic callus tissue was transferred to NB solid selection medium containing 50 mg / L hygromycin and incubated at 28ºC in the dark for 7-10 days. Afterward, it was subcultured into new NB solid selection medium containing hygromycin for 3-4 rounds of hygromycin selection. The callus tissue in good condition was propagated to differentiation medium for differentiation and regeneration, and seedlings were cultured at 28ºC for about one month. The seedlings were then transferred to rooting medium and cultured for about four weeks to promote rooting and strengthen the seedlings. An appropriate amount of distilled water was added, and the seedlings were hardened off for three days. The medium was washed off the roots of the seedlings, and they were moved to a greenhouse for about one month of growth. Finally, they were transplanted to an experimental field (located in Beijing, China, 116.2312 degrees east longitude, 40.2210 degrees north latitude) to obtain T0 generation plants.

[0075] 2. Identification of mutation sites in positive knockout plants The specific methods are as follows: (1) Leaves were taken from each of the above T0 generation transgenic rice plants, and rice genomic DNA was extracted by CTAB method as a template. PCR amplification was performed using OsWRKY31-F and OsWRKY31-R identification primers. The PCR products were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for Sanger sequencing. After obtaining the sequencing results, the sequences were compared. Those sequences that matched the expected sequences were T0 generation positive plants. A total of 12 T0 generation positive plants of rice and upland rice were obtained this time.

[0076] (2) Five positive T0 generation plants were randomly selected for propagation, i.e., five T1 generation lines were planted. Six individual plants were randomly collected from each T1 generation line, which can be used to screen for homozygous positive individual plants.

[0077] (3) The harvested T1 generation seeds were germinated. Forty germinated seeds were placed on 0.5% Agar solid medium containing 50 μg / mL hygromycin. Root growth was observed after 2-3 days. Plants with normal root growth were considered positive; otherwise, they were considered isolated pseudo-plants. If all seeds of each plant could grow roots normally, it was considered a homozygous positive line. Four homozygous positive plants were obtained from both paddy rice and upland rice.

[0078] Primers for identifying transgenic positive seedlings: OsWRKY31-F: 5'-CTTTCTTTCTTCTCTGAAAC -3'; OsWRKY31-R: 5'-ACTGAGTACAATGGTGGATC-3'.

[0079] PCR reaction system: 1µL DNA template, 2µL 10µM upstream primer OsWRKY31-F, 2µL 10µM downstream primer OsWRKY31-R, 25µL Novizan Rapid Taq Master Mix, 20µL ddH2O.

[0080] PCR reaction program: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 15 s, 35 cycles; 72℃ extension for 5 min.

[0081] Two homozygous mutant rice plants were obtained this time. Oswrky31-1 and Oswrky31-2 Compared with wild-type rice Nipponbare, the mutant plants showed better performance in terms of... OsWRKY31 The gene underwent the following mutation: in the mutant plant Oswrky31-1 middle OsWRKY31 The CDS coding sequence (SEQ ID NO:2) in the gene contains a 5-base deletion at position 205 (i.e., the deletion of nucleotides 734 to 738 of SEQ ID NO:3, ACGAA) and a 33-base deletion at position 323 (i.e., the deletion of nucleotides 852 to 884 of SEQ ID NO:3, AGACTGCATCTGGGAAGGCAGTCTCCATCTGCT). These nucleotide changes cause premature termination of protein translation. Figure 1 ).

[0082] Compared to wild-type Nipponbare rice, the mutant plants Oswrky31-2 middle WRKY31The CDS coding sequence in the gene has a 17-base deletion at position 205 (i.e., the deletion of nucleotides 734 to 750 of SEQ ID NO:3, ACGAAGCAGATTCAGCA) and a 1-base insertion at position 331 (i.e., the insertion of an A between nucleotides 859 and 860 of SEQ ID NO:3). These nucleotide changes cause premature termination of protein translation. Figure 1 ).

[0083] Two homozygous mutant plants of upland rice were obtained this time. Oswrky31-3 and Oswrky31-4 Compared with wild-type upland rice, mutant plants are more effective in... OsWRKY31 The gene underwent the following mutation: in the mutant plant Oswrky31-3 middle OsWRKY31 There is a 128-base deletion at position 204 of the CDS coding sequence in the gene (i.e., nucleotides 733 to 860 of SEQ ID NO:3). Figure 2 ).

[0084] Compared with wild-type dryland rice, in mutant plants Oswrky31-4 middle OsWRKY31 The CDS coding sequence in the gene contains an insertion of one base at position 206 (i.e., an A is inserted between nucleotides 734 and 735 of SEQ ID NO:3), and a deletion of two bases at position 329 (i.e., the deletion of nucleotides 858 and 859 of SEQ ID NO:3). These nucleotide changes cause premature termination of protein translation. Figure 2 ).

[0085] Example 3: Transformed rice and upland rice plants Oswrky31 Identification of drought-resistant phenotypes in mutants 1. Drought stress treatment experiment To detect rice transformation plants Oswrky31 / NP mutants and drought-resistant rice transformants Oswrky31 Compared to wild-type rice and upland rice, the drought resistance of / IRAT109 was compared with that of wild-type rice Nipponbare and wild-type upland rice IRAT109 with transformed plants. Oswrky31-1, Oswrky31-2, Oswrky31-3 and Oswrky31-4 The mutant seeds were soaked in 37℃ water for 2-3 days to promote germination. Seeds with the most consistent germination were then selected and planted in pots containing a 1:1 mixture of vermiculite and soil. Normal watering was maintained during the growth period until the four-leaf stage. Afterward, irrigation was stopped, and the four-leaf seedlings were subjected to a 15-day drought treatment, followed by a 5-day watering treatment. The growth status of wild-type and mutant seedlings was observed, and their survival rates were recorded.

[0086] Leaf moisture content detection method: One day before the end of drought treatment, select the second leaf from the top of the seedlings and immediately weigh the fresh weight of the leaves; then place the leaves in an oven to dry to constant weight, cool and weigh to obtain the dry weight. Leaf moisture content calculation formula: (fresh weight - dry weight) / fresh weight * 100%.

[0087] Figure 3 It is a mutant of wild-type rice Nipponbare and rice transformant plants. Oswrky31-1 and Oswrky31-2 The drought resistance phenotype (A), survival rate (B), and leaf water content (C) were measured in three biological replicates, with 16 rice seedlings per line per replicate. As shown in the figure, after 15 days of drought treatment and 5 days of rehydration treatment, the growth and development of the rice mutant seedlings were significantly better than those of the wild type. Regarding survival rate, the survival rate of wild-type Nipponbare rice was less than 30%, while that of the rice mutant seedlings was significantly higher. Oswrky31 The survival rate was approximately 75%, indicating that the rice mutant plants were transformed. Oswrky31 The survival rate was significantly higher than that of the wild type, and the difference between the two was extremely significant, with P < 0.01 (**) and P < 0.001 (***). Oswrky31 The leaf water content after drought was also significantly higher than that of the wild type (P < 0.001). Therefore, this rice mutant plant... Oswrky31 It can significantly improve drought resistance.

[0088] Figure 4 It is a mutant of wild-type upland rice IRAT109 and upland rice transformant plants. Oswrky31-3 and Oswrky31-4 The drought resistance phenotype (A), survival rate (B), and leaf water content (C) were measured in three biological replicates, with 16 rice seedlings per line in each replicate. As shown in the figure, after 18 days of drought treatment and 5 days of rehydration treatment, the growth and development of the mutant seedlings of the upland rice were significantly better than those of the wild type. Survival rates were statistically analyzed; the survival rate of the wild-type upland rice IRAT109 was approximately 10%, while that of the upland rice... Oswrky31 The survival rate of the mutants was approximately 50%–60%, indicating that the mutants of the drought-resistant rice transformed plants… Oswrky31 The survival rate was significantly higher than that of the wild type, and the difference between the two was extremely significant (P < 0.001). Oswrky31 The leaf water content after drought was also significantly higher than that of the wild type (P < 0.001). Therefore, this drought-transformed rice mutant... Oswrky31 It can significantly improve drought resistance.

[0089] 2. Detection of ROS content before and after drought treatment Drought stress typically leads to the accumulation of reactive oxygen species (ROS) in plants, including hydrogen peroxide and superoxide anions. These substances can cause oxidative damage to plant cells and even plant death. 3,3'-Diaminobenzidine (DAB) staining is a histochemical method for specifically detecting hydrogen peroxide (H₂O₂) in plant tissues. The principle of DAB staining is that H₂O₂ oxidizes DAB under the catalysis of peroxidase (POD), forming an insoluble brown polymer precipitate. The accumulation of this precipitate is positively correlated with the concentration of H₂O₂. Therefore, observing the staining depth allows for a semi-quantitative comparison of ROS levels between different samples.

[0090] The specific procedures for DAB staining to detect ROS are as follows: (1) Material preparation: Take leaves from the same developmental stage of the plant to be tested (such as the third fully expanded leaf after drought treatment); DAB solution (1 mg / mL DAB, dissolved in 10 mM phosphate buffer at pH 3.8, freshly prepared or stored in the dark), stop solution (anhydrous ethanol: glacial acetic acid: glycerol = 3:1:1), bleach (95% ethanol) and other reagents.

[0091] (2) Infiltration treatment: ① Immerse the leaves in DAB solution and vacuum (-0.1 MPa, 10 min) to promote reagent penetration. ② Transfer to a light incubator (25℃) and incubate in the dark for 6-8 h.

[0092] (3) Termination of reaction: Take out the leaves, rinse them three times with distilled water, and immerse them in the termination solution and boil for 10 minutes to inactivate the enzyme activity.

[0093] (4) Decolorization and observation: ① Transfer the leaves to 95% ethanol for decolorization, and bathe in a 60℃ water bath until the chlorophyll is completely removed. ② After rinsing with distilled water, photograph the leaves against a white background, or store them in glycerol at 4℃.

[0094] (5) Results analysis: The higher the density of brown bands, the more H2O2 accumulates.

[0095] Figure 5 To investigate the relationship between wild-type rice Nipponbare and rice transformant plants at the four-leaf stage Oswrky31 DAB staining of mutants before and after drought treatment. DAB staining was used to examine wild-type Nipponbare rice and rice transformants at the four-leaf stage. Oswrky31 ROS levels in mutants showed that after drought treatment Oswrky31 The mutant's H2O2 accumulation was significantly lower than that of wild-type Nipponbare rice, indicating that the transformed rice plants showed a greater reduction in H2O2 accumulation compared to the wild-type Nipponbare. Oswrky31 The mutants exhibit a significantly enhanced ability to cope with drought stress.

[0096] Figure 6To compare wild-type upland rice IRAT109 at the four-leaf stage with upland rice transformants Oswrky31 DAB staining of mutants after drought treatment. DAB staining was used to examine wild-type paddy rice IRAT109 and upland rice transformants at the four-leaf stage. Oswrky31 ROS levels in mutants showed that after drought treatment Oswrky31 The mutant's H2O2 accumulation was significantly lower than that of wild-type upland rice IRAT109, indicating that the transformed upland rice plants showed a greater reduction in H2O2 accumulation compared to wild-type IRAT109. Oswrky31 The mutants exhibit a significantly enhanced ability to cope with drought stress.

[0097] The sequences involved in the above embodiments OsWRKY31 The amino acid sequence of the gene-encoded protein (SEQ ID NO:1) MSPVPSPHQSHHLGHGSRKEKRMRKVDTFAPHNDGHQWRKYGEKKINNCNFPRYYYRCTYKDNMNCPATKQIQQKDYSDPPLYSVTYYNEHTCNSAFLPLSPSEF QLQTASGKAVSICFESSGAQEPMTNASSPSSSAARRSTPSENKNQPLPRHSEAYSWGVGVVEQKPSCTELQSCSTECQDAFSAGTIPEETVDAGRFGSIRFFHFL.

[0098] OsWRKY31 CDS sequence of the gene (SEQ ID NO:2) 5'-ATGTCTCCTGTGCCGAGTCCGCATCAATCACACCATCTAGGCCATGGCTCAAGGAAAGAGAAGCGCATGAGGAAGGTGGATACCTTTGCGCCGCACAACGACGGCCACCAGTGGAGGAAGTACGGCGAGAAGAAGATAAACAACTGTAATTTCCCCAGATACTACTACAGATGCACCTATAAAGATAACATGAATTGCCCAGCAACGAAGCAGATTCAGCAGAAAGATTATAGTGATCCACCATTGTACTCAGTCACCTACTACAATGAGCATACATGTAATAGTGCTTTTCTTCCTCTTAGCCCCTCAGAGTTCCAGCTGCAGACTGCATCTGGGAAGGCAGTCTCCATCTGCTTTGAATCATCTGGGGCTCAAGAACCAATGACCAATGCCAGCTCACCTTCTTCAAGCGCAGCACGGCGTAGCACACCTTCAGAGAACAAGAATCAGCCTCTTCCACGGCATTCGGAAGCCTATTCTTGGGGGGTTGGTGTTGTAGAACAAAAGCCGTCCTGCACTGAGCTCCAATCTTGCAGCACCGAATGTCAGGATGCATTTTCAGCTGGTACGATTCCTGAAGAGACAGTAGATGCAGGAAGATTTGGTTCTATCAGATTCTTCCATTTTTTGTAA-3'。

[0099] OsWRKY31 Genomic sequence of the gene (SEQ ID NO:3)

[0100] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application, characterized in that, The application includes the use of a protein or a substance that inhibits, reduces, or downregulates the expression of the protein-encoding gene or a substance that inhibits, reduces, or downregulates the activity or content of the protein in any of the following: C1) Improve the drought resistance of plants; C2) Prepare products that improve plant drought resistance; C3) Cultivate plants with improved drought resistance; C4) Prepare products from plants with improved drought resistance; C5) Plant breeding or assisted plant breeding; The protein is OsWRKY31, and OsWRKY31 is any of the following: A1) The amino acid sequence is that of the protein SEQ ID NO:1; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

2. The application according to claim 1, characterized in that, The substance is any one of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein of claim 1, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the protein, or RNA molecules that knock out the gene encoding the protein of claim 1. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7) Transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) A transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

3. A method for improving the drought resistance of plants, comprising inhibiting or reducing or downregulating the expression of the gene encoding the protein in the recipient plant or inhibiting or reducing or downregulating the activity and / or content of the protein, thereby improving the drought resistance of the recipient plant, wherein the recipient plant contains the gene encoding the protein.

4. The method according to claim 3, characterized in that, The inhibition, reduction, or downregulation of the expression of the gene encoding the protein of claim 1 in the recipient plant, or the inhibition, reduction, or downregulation of the protein activity and / or content, includes introducing into recipient rice a substance that inhibits, reduces, or downregulates the expression of the gene encoding the protein of claim 1 in the recipient plant, or inhibits, reduces, or downregulates the protein activity and / or content, or knocks out the gene encoding the protein of claim 1 in the recipient plant.

5. The method according to claim 4, characterized in that, The gene encoding the protein in the knockout recipient plant comprises a gene knockout vector into the recipient plant that targets the reverse complementary sequence of nucleotides 730-749 of SEQ ID NO:3 and / or nucleotides 843-862 of SEQ ID NO:

3.

6. A method for cultivating plants with enhanced drought resistance, wherein the expression of the gene encoding the protein described in claim 1 in the starting plant is inhibited, reduced, or downregulated, or the activity and / or content of the protein is regulated, to obtain a target plant, wherein the target plant has higher drought resistance than the starting plant.

7. The method according to claim 6, characterized in that, The inhibition, reduction, or downregulation of the expression of the gene encoding the protein of claim 1 in the starting plant, or the inhibition, reduction, or downregulation of the protein activity and / or content, includes introducing into the starting rice a gene that inhibits, reduces, or downregulates the expression of the gene encoding the protein of claim 1 in the starting plant, or inhibits, reduces, or downregulates the protein activity and / or content, or knocks out the gene encoding the protein of claim 1 in the starting plant.

8. The method according to claim 7, characterized in that, The gene encoding the protein in the knockout starting plant comprises introducing a gene knockout vector into the starting plant with the reverse complementary sequence of nucleotides 730-749 of SEQ ID NO:3 and / or nucleotides 843-862 of SEQ ID NO:3 as the target site.

9. The method according to claim 8, characterized in that, The gene encoding the target plant to be knocked out is obtained by mutating the gene encoding the target plant using one or more of the following methods: K1) Delete nucleotides 734 to 738 of SEQ ID NO:3; K2) Delete nucleotides 852 to 884 of SEQ ID NO:3; K3) Delete nucleotides 734 to 750 of SEQ ID NO:3; K4) An A is inserted between nucleotides 859 and 860 of SEQ ID NO:3; K5) Delete nucleotides 734 to 859 of SEQ ID NO:3; K6) An A is inserted between nucleotides 734 and 735 of SEQ ID NO:3; K7) Delete nucleotides 858 to 859 of SEQ ID NO:

3.

10. A biomaterial, characterized in that, The biomaterial is at least one of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein of claim 1, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the protein, or RNA molecules that knock out the gene encoding the protein of claim 1. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7) Transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) Transgenic plant organs containing the gene described in B2), or transgenic plant organs containing the expression cassette described in B3), or transgenic plant organs containing the recombinant vector described in B4); B9) A transgenic plant containing the gene described in B2), or a transgenic plant containing the expression cassette described in B3), or a transgenic plant containing the recombinant vector described in B4).

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