Application of OsWRKY26 protein in regulation and control of cold resistance of rice in seedling stage

Abstract

The invention relates to application of OsWRKY26 protein in regulation and control of cold resistance of rice in a seedling stage, belonging to the technical field of biology. The OsWRKY26 protein is a protein as shown in A1, A2 or A3: A1 is a protein with an amino acid sequence shown in SEQ ID No.1 in a sequence table; a2, a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues on the protein of A1, has 75% or more of identity with the protein shown in A1, and has the activity of regulating and controlling the cold tolerance of rice in the seedling stage; and A3, a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of A1 or A2.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a... OsWRKY Application of protein 26 in regulating cold tolerance in rice seedlings. Background Technology

[0002] Rice Sorghum bicolor Rice (L.) is one of the world's four major cereal crops. It is a warm-loving crop originating in tropical or subtropical regions, with good heat tolerance but extremely sensitive to low temperatures. In high-latitude and high-altitude low-temperature production areas, low-temperature damage during the seedling stage of rice has become one of the important factors affecting its yield and planting area.

[0003] Chilling injury, an agricultural meteorological disaster, can occur throughout the crop growing season. When it occurs, it typically causes varying degrees of damage to crops, affecting their normal growth and ultimately leading to a decline in quality and yield. In actual production, rice is highly susceptible to chilling injury, especially during its vegetative growth stage. The root cause lies in the fluctuating temperatures during early spring sowing, with frequent occurrences of low temperatures and late spring frosts. This causes rice seedlings to rot after emerging under low-temperature stress, resulting in later yield losses and severely restricting rice production.

[0004] Therefore, cold damage during the seedling stage is a critical period affecting rice yield, making research on cold tolerance in rice seedlings extremely important. However, developing cold-tolerant rice varieties using traditional breeding methods is a long process and progresses slowly. In contrast, genetic engineering technology can directly modify the genetic background of plants at the gene level, allowing for targeted modification of genetic traits. Discovering cold-tolerant genes in rice seedlings has significant theoretical and practical implications for developing new cold-tolerant rice varieties. Summary of the Invention

[0005] This invention is intended to provide OsWRKY Application of protein 26 in regulating cold tolerance in rice seedlings.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: OsWRKY Application of protein 26 in regulating cold tolerance in rice seedlings OsWRKY Protein 26 is a protein as shown in A1, A2, or A3: A1. The amino acid sequence is that of the protein listed as SEQ ID No. 1 in the sequence listing; A2, a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1, which has more than 75% identity with the protein shown in A1 and has the activity of regulating cold tolerance in rice seedlings. A3. A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1 or A2.

[0007] Amino acid sequence SEQ ID No. 1: MYMAAAAAGASTPFNFCRHGSHAEYDAVFSGSWMARRPSAAPHGGGASGSGSGSGYGAASYVAPTFGAAFRQQHLDLLDYLSDDQGVPAPPPAAVPSASYVTPAPAMAPAEPVVPDAVAAAGG YPRSVAAAAAAVAGEGRDRTTTDKIAFRTRSDDEILDDGYKWRKYGKKSVKNSPNPRNYYRCSTEGCNVKKRVERDKNDPRYVVTMYEGIHNHVCPGTVYYAAQDAASGRFFVAGISHPDLN.

[0008] In the above applications, protein identity refers to the identity of amino acid 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 program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0009] In the above applications, the 75% or more identity of the proteins can be at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity.

[0010] In the above applications, the protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc. The amino acid sequences of some available tags are shown in Table 1.

[0011] Table 1. Amino acid sequence of the tag

[0012] Preferably, as an improvement, the OsWRKY Protein 26 is derived from rice or synthesized artificially.

[0013] Preferably, as an improvement, the OsWRKY Protein 26 is encoded by a gene, the coding sequence of which is the cDNA molecule or DNA molecule of SEQ ID No. 2.

[0014] SEQ ID No. 2: .

[0015] Preferably, as an improvement, the application is as follows: OsWRKY Application of protein 26 in improving cold tolerance of rice seedlings.

[0016] Preferably, as an improvement, it promotes the [specific benefits] in recipient rice. OsWRKY26 protein expression and / or increase OsWRKY Increased protein abundance enhances cold tolerance in rice seedlings.

[0017] Preferably, as an improvement, the receptor-inhibiting rice described OsWRKY 26 protein expression, reduction OsWRKY 26. Abundance and / or knockout encoding of the protein described OsWRKY The gene for protein 26 reduces the cold tolerance of rice seedlings.

[0018] Preferably, as an improvement, the receptor-inhibiting rice described OsWRKY 26 protein expression, reduction OsWRKY 26. Abundance and / or knockout encoding of the protein described OsWRKY The gene for protein 26 was expressed using a CRISPR / Cas9 system, which includes a plasmid expressing Cas9 and sgRNA, the target sequence of which is positions 193-212 in the sequence listing of SEQ ID No. 2.

[0019] Preferably, as an improvement, the OsWRKY The expression of protein 26 is regulated by a gene, which is regulated by any one of the following F1, F2, and F3 substances: F1, sgRNA, siRNA, shRNA, miRNA, or antisense RNA targeting the gene; F2. Generate DNA molecules that target the gene using sgRNA, siRNA, shRNA, miRNA, or antisense RNA. F3. Generate expression vectors that target the gene using sgRNA, siRNA, shRNA, miRNA, or antisense RNA.

[0020] Preferably, as an improvement, the receptor in rice is inhibited by a reagent. OsWRKY 26 protein expression and / or reduction OsWRKY 26. The abundance of protein, and the active ingredient of the reagent is the substance described.

[0021] This invention provides the application of a substance that regulates gene expression in regulating cold tolerance in rice seedlings or in the preparation of products that regulate cold tolerance in rice seedlings, wherein the gene encodes... OsWRKY 26 protein. This invention uses gene editing technology to knock out rice... OsWRKY26 This invention provides a foundation for obtaining new rice germplasm with significantly improved cold tolerance during the seedling stage. It is of great significance for the innovation of cold-tolerant rice germplasm resources, the breeding of new varieties, environmental sanitation, and food security, and has significant application and promotion value. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of a climate chamber for seedling material treatment in an embodiment of the present invention.

[0023] Figure 2 The figures show the statistical results of the seedling phenotypes and cold tolerance-related physiological indicators of two rice varieties, Kunmingxiaobaigu (KMXBG) and Nipponbare (Nip), after 7 days of low-temperature treatment in Example 1 of this invention. Figure a shows the seedling photos of the two materials after low-temperature treatment (right side) and normal control (left side). Figures b, c, d, and e are bar charts of seedling survival rate, chlorophyll content, ion permeability, and proline content, respectively. In the figures, "**" indicates p < 0.01, "***" indicates p < 0.001, and ns indicates no significant difference.

[0024] Figure 3 In Embodiment 1 of the present invention OsWRKY26 Results of heatmap analysis of genes.

[0025] Figure 4 In Embodiment 1 of the present invention OsWRKY26 Expression results in different tissues of KMXBG and Nip, with internal references as follows: OsActin1 Gene.

[0026] Figure 5 In Embodiment 1 of the present invention OsWRKY26 Expression pattern analysis of KMXBG and Nip seedlings under cold induction, with internal reference as... OsActin1 Gene.

[0027] Figure 6 In Embodiment 2 of the present invention OsWRKY26 Results of phenotype, gene expression level, seedling survival rate and related cold tolerance indicators of knockout strains; Figures a, b, c, d, e, f and g are statistical graphs of phenotype, gene expression level, seedling survival rate, chlorophyll content, ion permeability, proline content and malondialdehyde content, respectively; different lowercase letters in the figures indicate significant differences in seed setting rate among different varieties.

[0028] Figure 7 In Embodiment 2 of the present invention OsWRKY26 Results of gene expression levels, seedling survival rate and related cold tolerance indicators of rice overexpression lines and control plants after cold treatment; Figures a, b, c, d, e, f and g are statistical graphs of phenotype, gene expression level, seedling survival rate, chlorophyll content, ion permeability, proline content and malondialdehyde content, respectively. Detailed Implementation

[0029] 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.

[0030] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0032] The rice varieties KMXBG and Nip in the following examples are both local materials. The original species are preserved at the College of Agriculture of China Agricultural University. The public can obtain them from Maotai College (i.e., the applicant) to repeat the experiments of this application.

[0033] The Agrobacterium tumefaciens strain EHA105 in the following examples is described in the non-patent literature “Gao Shiwu et al. Study on factors affecting the transformation efficiency of Agrobacterium tumefaciens EHA105 competent cells. Journal of Tropical Biology. March 2012, Vol. 3, No. 1”, which can be obtained by the public from Maotai College (i.e. the applicant) to replicate the experiments of this application.

[0034] The following examples use Excel statistical software to process the data. The experimental results are expressed as mean ± standard deviation and Duncant's test is used. "*" indicates p<0.05, "**" indicates p<0.01, and "***" indicates p<0.001.

[0035] Example 1 OsWRKY26 Acquisition of genes 1. OsWRKY26 Acquisition of genes In this study, two rice varieties with significant differences in cold tolerance during the seedling stage were selected: Kunmingxiaobaigu (KMXBG) and Nipponbare (Nip).

[0036] The method for determining cold resistance is as follows: Artificial Climate Chamber (ZRX-460B) Cold Treatment Figure 1 ): Rice seedlings that have grown to the seedling stage (three-leaf stage) under normal conditions are transferred to an artificial climate chamber and treated at 4℃ for 7 days.

[0037] Control: Rice grown at room temperature to the seedling stage under normal conditions was not subjected to low temperature stress.

[0038] 14 plants per material, repeated 3 times.

[0039] After the cold stress ended, the tested materials were transferred to room temperature to recover growth. One week later, phenotypic identification was performed, and seedling survival rate was recorded. Seedling survival rate = (Number of surviving seedlings after treatment / Total number of seedlings) × 100% Phenotypic identification results are shown in Figure 2 This indicates that KMXBG seedlings have significantly higher cold tolerance than Nip seedlings. Two rice varieties that had grown to the seedling stage under normal conditions were transferred to an artificial climate chamber at 4°C for 7 days of treatment, with the control group receiving 0 hours of treatment. Transcriptome sequencing was performed on panicles at 0, 2, 8, and 16 hours of low-temperature treatment to obtain a series of differentially expressed genes. Comparative analysis of transcriptome factors among these differentially expressed genes identified five genes shared across different treatment time points, and a heatmap analysis of these five genes was performed based on log2FC values. Figure 3 ).Discover LOC_Os01g51690 The gene was induced to express at low temperatures in both rice materials, with a higher degree of induction in the cold-tolerant material KMXBG than in the cold-sensitive material NIP. Furthermore, the upregulation of this gene's expression in KMXBG was induced by low temperatures earlier than in NIP. Therefore, it is speculated that... LOC_OS01G51690 It may be a candidate gene for regulating cold tolerance in rice seedlings. LOC_OS01G51690 The nucleotide sequence of the gene is SEQ ID No. 2, and the protein it encodes is... OsWRKY 26. The amino acid sequence of this protein is SEQ ID No. 1.

[0040] 2. OsWRKY26 Gene expression patterns Normal processing: Take samples from different tissues under normal Nip growing conditions, including seedlings, roots, stems, mature leaves, leaf sheaths, and spikelets. After sampling, flash freeze in liquid nitrogen and store at -80℃ for later use.

[0041] Low temperature stress treatment: KMXBG and NIP seedlings grown to the seedling stage under normal conditions were transplanted into an artificial climate chamber at 4℃. Leaf samples were taken from the seedlings at 0h, 2h, 8h, 16h, 2d, 4d and 6d after treatment, respectively, and were flash-frozen with liquid nitrogen and stored at -80℃ for later use.

[0042] Total RNA was extracted from samples treated in the above ways, and the first strand of cDNA was synthesized using reverse transcriptase M-MLV. Using this first strand of cDNA as a template, real-time quantitative analysis was performed using primers. OsActin1 It is an internal reference gene. OsWRKY26 The amplification primer set consists of qRT- OsWRKY 26-F and qRT- OsWRKY Composed of 26-R, OsActin1 The amplification primer set consists of Actin1-F and Actin1-R. The sequences are as follows: OsWRKY26 Amplification primers: qRT- OsWRKY 26-F: 5'- GACGGGTATAAGTGGAGGAAGTAC-3'; qRT- OsWRKY 26-R: 5'-CGTCGAGCACCGGTAGTAG-3' OsActin1 Amplification primers: Actin1-F: 5'-CGAAGACATACTGGACGCAACA-3'; Actin1-R: 5'-GGGCGGAAAGAATTAGAAGC-3'; The experiment was repeated three times.

[0043] OsWRKY26 Organizational expression patterns, such as Figure 4 It can be seen that OsWRKY26 A gene is a gene that is expressed in different tissues, especially in the ear and seedling.

[0044] OsWRKY26 Cold-induced expression analysis, such as Figure 5 It can be seen that both KMXBG and NIP are induced by low temperature, indicating that... OsWRKY26 The gene is one that is associated with low temperature stress.

[0045] Example 2 OsWRKY26 Functional verification of genes 1. OsWRKY26 Construction of knockout vector The target was obtained from the website https: / / crispor.gi.ucsc.edu / , and the specific target sequence is the nucleotide sequence of positions 193-212 bp of SEQ ID No. 2 (a total of 20 bp).

[0046] The coding sequence of sgRNA is: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC.

[0047] Four-primer PCR amplification was performed using the vector pCBC-MT1T2 diluted 100-fold as a template. The primers used are as follows: OsWRKY 26-MT1-BsF:5-ATATATGGTCTCTGGCGACCTTCGGCGCGGCGTTTAGGTT-3'; OsWRKY 26-MT1-F0:5-TGACCTTCGGCGCGGCGTTTAGGTTTTAGAGCTAGAAATAGC-3'; OsWRKY 26-MT2-R0:5-AACCGTCCGGAATGCGATCTTGTGCTTCTTGGTGCC-3'; OsWRKY 26-MT2-BsR:5-ATTATTGGTCTCTAAACCGTCCGGAATGCGATCTTGT-3'; OsWRKY 26-MT1-BsF / OsWRKY 26-MT2-BsR is the normal primer concentration; OsWRKY 26-MT1-F0 / OsWRKY 26-MT2-R0 diluted 20 times.

[0048] The PCR product was obtained by four-primer PCR amplification and purified. Using BsaI as the restriction enzyme site, the purified PCR product was subjected to enzyme digestion-ligation reaction according to the following steps: 2 μL of purified PCR product, 2 μL of vector pBUE411, 1.5 μL of 10×NEB T4 Buffer, 1.5 μL of 10×Caster Buffer, 2 μL of BsaI (NEB), 1 μL of T4 Ligase (NEB), and 6 μL of ddH2O. The reaction was carried out at 37℃ for 5 h, 50℃ for 5 min, and 80℃ for 10 min. PCR single-clone detection was performed (using OsU3-FD3 and TaU3-RD as the detection primer pair, with a target band size of 831 bp), and sequencing was performed using primers OsU3-FD3 and TaU3-FD2. The specific primer sequences are as follows: OsU3-FD3:5'-GACAGGCGTCTTCTACTGGTGCTAC-3'; TaU3-RD:5'- CTCACAAATTATCAGCACGCTAGTC-3'; TaU3-FD:5'-TTAGTCCCACCTCGCCAGTTTACAG-3'; TaU3-FD2:5'-TTGACTAGCGTGCTGATAATTTGTG-3'.

[0049] Sequencing results confirmed the presence of [something] on the obtained recombinant vector. OsWRKY 26 knockout target, finally obtained OsWRKYThe site-directed knockout vector for 26 genes was named pBUE411- OsWRKY 26.

[0050] 2. OsWRKY 26. Obtaining genetically modified rice by knocking out genetically modified organisms The above-prepared material with OsWRKY 26. Recombinant vector pBUE411- with target knockout OsWRKY 26. Recombinant strain EHA105 was obtained by transforming Agrobacterium tumefaciens EHA105 using the freeze-thaw method. OsWRKY 26, used to infect callus of transgenic receptor Nip.

[0051] Nip was infected using the classic Agrobacterium-mediated callus infection method to obtain transgenic rice. Genomic DNA was extracted and amplified using primer pairs composed of F and R. F: 5'-ATGTACATGGCGGCGGCG-3'; R: 5'-TTAATTAAGGTCTGGGTG-3'.

[0052] A 738 bp PCR product was amplified and sequenced with wild-type Nip. Positive T0 transduction was indicated by insertions, deletions, or substitutions of nucleotides at or near the target site. OsWRKY 26. Rice plants with no sequence changes were negative.

[0053] Positive T0 plants were identified, and seeds were planted and harvested to obtain T1 generation seeds. This process was repeated until the T3 generation was obtained. OsWRKY 26 rice lines. Extraction of T3 transgenic strains. OsWRKY Genomic DNA from 26 rice lines was amplified using the F and R primer pairs described above, yielding homozygous T3 knockout lines. Phenotypic identification results are as follows: Figure 6 As shown ( OsWRKY26-2 , OsWRKY26-8 ).

[0054] strain OsWRKY26-2 For in genes OsWRKY In the nucleotide sequence SEQ ID No. 2, a nucleotide C or A was inserted between positions 195 and 196, resulting in the encoding protein. OsWRKY A frameshift occurred at 26:00, preventing the encoding of the protein. OsWRKY 26, obtained OsWRKY 26-knockout homozygous line.

[0055] strain OsWRKY26-8 For in genes OsWRKY The nucleotide sequence of 26 in SEQ ID No. 2 has a deletion of 3 GTA bases inserted between bases 201-202, resulting in the encoding of a protein.OsWRKY A frameshift occurred at 26:00, preventing the encoding of the protein. OsWRKY 26, obtained OsWRKY 26-knockout homozygous line.

[0056] The pure T3 generation was subsequently selected. OsWRKY 26 rice lines OsWRKY26-2 and OsWRKY26-8 Conduct cold resistance testing.

[0057] 3. OsWRKY26 Identification of cold tolerance in rice The method for determining cold resistance using an artificial climate chamber ( Figure 1 Wild-type rice NIP, OsWRKY26 Rice knockout lines OsWRKY26-2 and OsWRKY26-8 Seedlings (three-leaf stage) were transferred to an artificial climate chamber and treated at 4°C for 7 days. Each material was treated with 14 plants, and the treatment was repeated three times.

[0058] After the stress ended, the tested materials were transferred to room temperature to allow rice growth to resume. One week later, the seedling survival rate was observed and tallied. Survival rate = (Number of survivors after treatment / Total number) × 100% The statistical results of seedling survival rate and related cold tolerance indicators are as follows: Figure 6 As shown, it can be seen that under low temperature stress, the knockout strain ( OsWRKY26-2 , OsWRKY26-8 The seedling survival rate, chlorophyll and proline content of the WT seedlings were significantly lower than those of the control plants, while the ion permeability and malondialdehyde content were significantly higher than those of the control plants. Conversely, the results were as follows: Figure 7 As shown, overexpression in rice revealed that the seedling survival rate, chlorophyll and proline content of the overexpressing lines (OE2, OE6) under low temperature stress were significantly higher than those of the control plants, while the ion permeability and malondialdehyde content were significantly lower than those of the control plants.

[0059] The above results indicate that low temperatures during the seedling stage affect the survival rate of rice by influencing the levels of physiological indicators such as chlorophyll, proline, malondialdehyde, and ion permeability. Under low temperature stress, the survival rate of rice seedlings is related to... OsWRKY26 The expression levels of genes are positively correlated, indicating that genes... OsWRKY26 It has the function of positively regulating seedling tolerance and can reduce the impact of temperature on survival rate under cold stress.

[0060] The present invention has been described in detail above. For those skilled in the art, 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. Although specific embodiments have been given, 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. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. OsWRKY The application of protein 26 in regulating cold tolerance in rice seedlings is characterized by: OsWRKY Protein 26 is a protein as shown in A1, A2, or A3: A1. The amino acid sequence is that of the protein listed as SEQ ID No. 1 in the sequence listing; A2, a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1, which has more than 75% identity with the protein shown in A1 and has the activity of regulating cold tolerance in rice seedlings. A3. A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1 or A2.

2. As described in claim 1 OsWRKY The application of protein 26 in regulating cold tolerance in rice seedlings is characterized by: The OsWRKY Protein 26 is derived from rice or synthesized artificially.

3. The method according to claim 2 OsWRKY The application of protein 26 in regulating cold tolerance in rice seedlings is characterized by: The OsWRKY Protein 26 is encoded by a gene, the coding sequence of which is the cDNA molecule or DNA molecule of SEQ ID No.

2.

4. The method according to any one of claims 1 to 3 OsWRKY The application of protein 26 in regulating cold tolerance in rice seedlings is characterized by: OsWRKY Application of 26 protein in improving cold tolerance of rice seedlings.

5. The method according to claim 4 OsWRKY The application of protein 26 in regulating cold tolerance in rice seedlings is characterized by: Promotes the receptor rice described OsWRKY 26 protein expression and / or increase OsWRKY Increased protein abundance enhances cold tolerance in rice seedlings.

6. The method according to claim 3 OsWRKY The application of protein 26 in regulating cold tolerance in rice seedlings is characterized by: The inhibitory receptor rice described OsWRKY 26 protein expression, reduction OsWRKY 26. Abundance and / or knockout encoding of the protein described OsWRKY The gene for protein 26 reduces the cold tolerance of rice seedlings.

7. The method according to claim 6 OsWRKY The application of protein 26 in regulating cold tolerance in rice seedlings is characterized by: The inhibitory receptor rice described OsWRKY 26 protein expression, reduction OsWRKY 26. Abundance and / or knockout encoding of the protein described OsWRKY The gene for protein 26 was expressed using a CRISPR / Cas9 system, which includes a plasmid expressing Cas9 and sgRNA, the target sequence of which is positions 193-212 in the sequence listing of SEQ ID No.

2.

8. The method according to claim 6 OsWRKY The application of protein 26 in regulating cold tolerance in rice seedlings is characterized by: The OsWRKY The expression of protein 26 is regulated by a gene, which is regulated by any one of the following F1, F2, and F3 substances: F1, sgRNA, siRNA, shRNA, miRNA, or antisense RNA targeting the gene; F2. Generate DNA molecules that target the gene using sgRNA, siRNA, shRNA, miRNA, or antisense RNA. F3. Generate expression vectors that target the gene using sgRNA, siRNA, shRNA, miRNA, or antisense RNA.

9. The method according to claim 8 OsWRKY The application of protein 26 in regulating cold tolerance in rice seedlings is characterized by: The receptor in rice was inhibited by a reagent. OsWRKY 26 protein expression and / or reduction OsWRKY 26. The abundance of protein, and the active ingredient of the reagent is the substance described.

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