Function and application of rice seedling cold tolerance related protein OsWRKY108

Abstract

The application discloses a protein OsWRKY108 for regulating cold tolerance of rice seedlings and an encoding gene and application thereof, an amino acid sequence of the OsWRKY108 protein is SEQ ID No. 1, or a variant with more than 75% identity with SEQ ID No. 1 and with the activity of regulating cold tolerance of rice seedlings, or a fusion protein with a protein tag connected to the N terminal and / or C terminal of the OsWRKY108 protein; and a nucleotide sequence of the encoding gene is SEQ ID No. 2. The application also discloses a substance for regulating expression of the gene, including sgRNA, siRNA, shRNA, miRNA or antisense RNA targeting the gene and an encoding carrier thereof. By inhibiting expression of the OsWRKY108 protein in a receptor rice, reducing the abundance or knocking out the encoding gene, the cold tolerance of the rice seedlings can be significantly improved; and overexpression of the gene reduces the cold tolerance. The application determines the function of the OsWRKY108 gene in the regulation of the cold tolerance of the rice seedlings, provides a key target and technical means for cultivating a new cold-tolerant rice variety, and has important theoretical and practical values for improving the rice yield and expanding the planting area.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the function and application of OsWRKY108, a protein related to cold tolerance in rice seedlings. Background Technology

[0002] Rice Sorghumbicolor Rice (L.) is one of the world's four major cereal crops. Originating in tropical or subtropical regions, it is a warm-climate crop that is extremely sensitive to low temperatures. In high-latitude and high-altitude areas with low temperatures, chilling injury during the seedling stage has become a significant factor affecting yield and planting area. Severe low-temperature stress during the seedling stage delays seedling growth, reduces seedling survival rate, and ultimately impacts rice yield.

[0003] Rice, an important food crop, originated in tropical or subtropical regions. It is a warm-season crop with good heat tolerance, but is highly sensitive to low-temperature chilling injury. Chilling injury, an agricultural meteorological disaster, can occur throughout the crop growing season. When it occurs, it usually causes varying degrees of damage to the crop, affecting its normal growth and ultimately leading to a decline in quality and yield. In actual production, rice is extremely susceptible to low-temperature 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 aims to provide the function and application of OsWRKY108, a protein related to cold tolerance in rice seedlings. By inhibiting the expression of OsWRKY108 protein in receptor rice, reducing its abundance, or knocking out its encoding gene, the cold tolerance of rice seedlings can be significantly improved; overexpression of this gene reduces cold tolerance. The function of the OsWRKY108 gene in regulating cold tolerance in rice seedlings has also been clarified, providing key targets and technical means for breeding new cold-resistant rice varieties.

[0006] 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 the OsWRKY108 protein, and the OsWRKY108 protein is a protein as follows (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) that 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] SEQ ID No.1 consists of 245 amino acid residues.

[0008] The above-mentioned proteins can be derived from rice.

[0009] 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.

[0010] 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. 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. Sequence of Labels

[0012] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0013] In the above applications, the gene is a nucleic acid molecule encoding the OsWRKY108 protein.

[0014] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0015] In the above applications, the gene may be a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No. 2.

[0016] In the above applications, the substance regulating gene expression is any one of the following F1, F2, or F3: F1, sgRNA, siRNA, shRNA, miRNA, or antisense RNA targeting the gene; F2. Generate a DNA molecule that targets the gene with sgRNA, a DNA molecule that targets the gene with siRNA, a DNA molecule that targets the gene with shRNA, a DNA molecule that targets the gene with miRNA, or a DNA molecule that targets the gene with antisense RNA. F3, generating an expression vector for sgRNA targeting the gene, generating an expression vector for siRNA targeting the gene, generating an expression vector for shRNA targeting the gene, generating an expression vector for miRNA targeting the gene, or generating an expression vector for antisense RNA targeting the gene.

[0017] To address the aforementioned technical problems, this invention provides a reagent for improving the cold tolerance of rice seedlings, wherein the active ingredient of the reagent is the aforementioned substance that regulates gene expression.

[0018] The active ingredients of the above reagents may also contain other biological and / or non-biological components. The other active ingredients of the above reagents can be determined by those skilled in the art based on the cold resistance effect of rice seedlings.

[0019] To address the aforementioned technical problems, the present invention also provides a method for improving the cold tolerance of rice seedlings, comprising the following steps: inhibiting the expression of the OsWRKY108 protein in recipient rice, reducing the abundance of the OsWRKY108 protein, and / or knocking out the gene encoding the OsWRKY108 protein, thereby obtaining target rice with higher cold tolerance during the seedling stage than the recipient rice.

[0020] In the above method, the inhibition of the expression of the OsWRKY108 protein in the receptor rice, the reduction of the abundance of the OsWRKY108 protein, and / or the knockout of the gene encoding the OsWRKY108 protein are achieved by a CRISPR / Cas9 system. The CRISPR / Cas9 system includes expressing a plasmid containing Cas9 and sgRNA. The target sequence of the sgRNA may be positions 63-82 or 392-411 of SEQ ID No. 2 in the sequence listing.

[0021] The present invention also provides the OsWRKY108 protein.

[0022] The present invention also provides biomaterials related to the OsWRKY108 protein, said biomaterials being any one of B1) to B5) below: B1) The nucleic acid molecule that encodes the protein; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the expression cassette described in B2).

[0023] This invention provides the application of a substance regulating 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 the OsWRKY108 protein. This invention uses gene editing technology to knock out the rice OsWRKY108 gene, providing a foundation for obtaining new rice germplasm with significantly improved cold tolerance in the seedling stage. This invention 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

[0024] Figure 1 This is a schematic diagram of a climate chamber for seedling material treatment in an embodiment of the present invention; Figure 2 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 are presented. Figure 2Figure a shows seedling photos of two materials after low-temperature treatment (right side) and normal control (left side). Figure 2 Figures b, c, d, and e are bar charts for seedling survival rate, chlorophyll content, ion permeability, and proline content, respectively. In the figures, "**" and "***" indicate significant differences, and ns indicate no significant differences. Figure 3 The VEEN analysis results of OsWRKY108 in Example 1 of this invention ( Figure 3 (A, 3B, 3C) and heatmap analysis results ( Figure 3 (D).

[0025] Figure 4 The results of OsWRKY108 expression in different tissues of Nip in Example 1 of this invention are shown, with the OsActin1 gene as the internal reference.

[0026] Figure 5 This is a diagram showing the expression pattern of OsWRKY108 under cold induction in KMXBG and Nip seedlings in Example 1 of the present invention. The internal reference is the OsActin1 gene.

[0027] Figure 6 This document presents the phenotypic, gene expression levels, seedling survival rate, and related cold tolerance indicators of the OsWRKY108 knockout strain in Example 2 of this invention. Figures a, b, c, d, e, f, and g show statistical charts for phenotypic, gene expression levels, 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 rates among different varieties.

[0028] Figure 7 The results show the gene expression levels, seedling survival rates, and related cold tolerance indicators of the OsWRKY108 rice overexpression line and control plants after cold treatment in Example 2 of this invention. Figure 7 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 following detailed description illustrates the specific implementation method: In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The following examples used Excel statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and Duncant's test was used. ，P＜0.05 (*) indicates a significant difference. P＜0.01 (**) indicates a highly significant difference.

[0034] Example 1 OsWRKY108 Acquisition of genes 1. Obtaining the OsWRKY108 gene In this study, two rice materials with significant differences in cold tolerance during the seedling stage were selected: Kunming Xiaobaigu (hereinafter referred to as KMXBG) and Nipponbare (hereinafter referred to as NIP).

[0035] Cold resistance identification method ( Figure 1 Specifically: Artificial climate chamber (ZRX-460B) cold treatment: Rice seedlings that have grown to the seedling stage (three-leaf stage) under normal conditions are transferred to the artificial climate chamber and treated at 4℃ for 7 days.

[0036] Control: Rice grown at room temperature to the seedling stage under normal conditions was not subjected to low temperature stress.

[0037] Fifteen plants were used for each material, and the experiment was repeated three times.

[0038] 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 grown to the seedling stage under normal conditions were transferred to an artificial climate chamber at 4℃ for 7 days of treatment, with a control group treated for 0 hours. Transcriptome sequencing was performed on panicles at 0h, 2h, 8h, and 16h of low-temperature treatment to obtain a series of differentially expressed genes. Venn comparative analysis was performed on the transcription factors among these differentially expressed genes. Figure 3 From A and B, five genes were identified that were common across different treatment time points, and a heatmap analysis was performed on these five genes based on the log2FC value. Figure 3 (D). Discovery of known functional genes. LOC_Os01g60600 ( OsWRKY108 The gene was induced by low temperature in both rice materials, and the induction level was higher in the cold-resistant material KMXBG than in the cold-sensitive material NIP. Furthermore, the upregulation of this gene by low temperature in KMXBG occurred earlier than in NIP. Studies have shown... OsWRKY108 It is a comprehensive regulator of phosphorus homeostasis and leaf angle in rice (Zhang et al. 2021; Wang et al., 2021), but whether it participates in the regulation of cold tolerance in rice is still unclear. Therefore, we speculate... OsWRKY108 It may be a candidate gene for regulating cold tolerance in rice seedlings. LOC_Os01g60600 The nucleotide sequence of the gene is SEQ ID No.2, and the protein it encodes is OsWRKY108, the amino acid sequence of which is SEQ ID No.1.

[0039] SEQ ID No. 1 (amino acid sequence) MQAQSRLAAAASGGSGSGISGSGGISRLGGGAGEEHEAVVRELTRGHELTARLRAEALRALRGQGQAEATATFILGEVSRAFTVCLSIMASASPSASPPQPDETPPADSAVSPPPPRAAREDNVPRKRLLTASPYDDGYQWRKYGQKKINNTNFPRSYYRCSYHRERRCPAQKHVQQRD GDDVPALHVVVYTHEHTCLQGAPAELPDAATNGGAAAAASPDYFPAGGETPSSLRRLRGVGGGGLQPQFVDHRAAMEERERQVLVSSLARVLQGRQCYDDDDDDTDVASLGAVHARAPAAAAPVAASSSSSGPVDAAGEELDVMDYDMTDALFWGPFGTDSNSYDGNLTSTRCFDLIN; SEQ ID No. 2 (nucleic acid sequence)

[0040] 2. OsWRKY108 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. SbActin1 It is an internal reference gene. OsWRKY108 The amplification primer set consists of qRT-OsWRKY108-F and qRT-OsWRKY108-R. OsActin1 The amplification primer set consists of Actin1-F and Actin1-R. The sequences are as follows: OsWRKY108 Amplification primers: qRT-OsWRKY108-F: 5'-CGTCCCGCGAAAGAGATTATTGAC-3'; qRT-OsWRKY108-R: 5'- CAACAACACCAACTTCCCAAGG-3' OsActin1 Amplification primers: Actin1-F: 5'-CGAAGACATACTGGACGCAACA-3'; Actin1-R: 5'-GGGCGGAAAGAATTAGAAGC-3' The experiment was repeated three times.

[0043] OsWRKY108 Organizational expression patterns, such as Figure 4 It can be seen that OsWRKY108 A gene is a gene that is expressed in different tissues, especially in the ear and seedling.

[0044] OsWRKY108 Cold-induced expression analysis, such as Figure 5 It can be seen that both KMXBG and NIP are subjected to low-temperature induction, with a higher degree of low-temperature induction in KMXBG, indicating that... OsWRKY108 The gene is one that is associated with low temperature stress.

[0045] Example 2 OsWRKY108 Functional verification of genes 1. OsWRKY108 Construction of knockout vector Through the website The target was obtained from https: / / crispor.gi.ucsc.edu / , and the specific target sequence is SEQ ID. No. 2, positions 63-82 or 392-411 nucleotide sequence.

[0046] Four-primer PCR amplification was performed using the vector pCBC-MT1T2 diluted 100-fold as a template. The primers used are as follows: OsWRKY108-MT1-BsF:5-ATATATGGTCTCTGGCGGAGCGGTGGCATTAGCAGGTGTT-3'; OsWRKY108-MT1-F0:5-TGGAGCGGTGGCATTAGCAGGTGTTTTAGAGCTAGAAATAGC-3'; OsWRKY108-MT2-R0:5-AACATCGTCGTAAGGAGAGGCTGGCTTCTTGGTGCC-3'; OsWRKY108-MT2-BsR:5-ATTATTGGTCTCTAAACATCGTCGTAAGGAGAGGCTG-3'; OsWRKY108-MT1-BsF / OsWRKY108-MT2-BsR are at normal primer concentrations; OsWRKY108-MT1-F0 / OsWRKY108-MT2-R0 are diluted 20-fold.

[0047] 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-clonal detection was performed (using OsU3-FD3 and TaU3-RD as the detection primer pair, with a target band size of 831 bp), and primers OsU3-FD3 and TaU3-FD2 were sequenced. 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'.

[0048] Sequencing results confirmed the presence of the OsWRKY108 knockout target on the obtained recombinant vector, and the OsWRKY108 gene knockout vector was finally obtained and named pBUE411-OsWRKY108.

[0049] 2. Obtaining OsWRKY108 knockout transgenic rice The recombinant vector pBUE411-OsWRKY108 with the OsWRKY108 knockout target prepared above was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant bacteria EHA105 / pBUE411-OsWRKY108, which was used to infect callus of transgenic receptor Nip.

[0050] 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'-ATGCAGGCGCAATCCCGCCT-3'; R: 5'-TTAATTAATTAGATCAAAACA-3'.

[0051] A PCR product of 1077 bp was amplified and compared with the wild-type Nip. Plants with insertions, deletions or substitutions in or near the target site were positive for T0 generation transgenic OsWRKY108 rice, while plants with no sequence changes were negative.

[0052] Positive T0 plants were identified and planted to obtain T1 generation seeds. These were then continuously self-crossed until T3 generation OsWRKY108 transgenic rice lines were obtained. Genomic DNA was extracted from the T3 generation OsWRKY108 transgenic rice lines and amplified using the F and R primer pairs described above. Homozygous T3 knockout lines were obtained. The identification results of some samples are shown below. Figure 6 As shown ( OsWRKY108-1 , OsWRKY108-5 ).

[0053] strain OsWRKY108-1The OsWRKY108 knockout homozygous line was obtained by inserting a nucleotide G between positions 83 and 84 in the nucleotide sequence SEQ ID No.2 of the OsWRKY108 gene, which caused a frameshift that prevented the encoding of the OsWRKY108 protein.

[0054] strain OsWRKY108-5 The OsWRKY108 knockout homozygous line was obtained by deleting three CCA bases between positions 405 and 406 in the nucleotide sequence of the OsWRKY108 gene (SEQ ID No. 2), causing a frameshift that prevents the encoding of the OsWRKY108 protein.

[0055] Subsequently, homozygous T3 generation transgenic OsWRKY108 rice lines were selected. OsWRKY108-1 and OsWRKY108-5 Conduct cold resistance testing.

[0056] 3. OsWRKY108 Identification of cold tolerance in rice The method for determining cold resistance using an artificial climate chamber ( Figure 1 Wild-type rice NIP, OsWRKY108 Rice knockout lines OsWRKY108-1 and OsWRKY108-5 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.

[0057] 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 ( OsWRKY108-1 , OsWRKY108-5 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, OE9) under low temperature stress were significantly higher than those of the control plants (WT), while the ion permeability and malondialdehyde content were significantly lower than those of the control plants.

[0058] 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... OsWRKY108The expression levels of genes are positively correlated, indicating that genes... OsWRKY108 It has the function of positively regulating seedling tolerance and can reduce the impact of temperature on survival rate under cold stress.

[0059] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention. These modifications and improvements should also be considered within the scope of protection of this invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A protein, OsWRKY108, that regulates cold tolerance in rice seedlings, characterized in that, The proteins shown in A1), A2), or A3) below: A1) A protein with the amino acid sequence SEQ ID No. 1; A2) is a protein obtained by substituting and / or adding and / or deleting amino acid residues from the amino acid sequence of A1), which has more than 75% identity with 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. A nucleic acid molecule encoding the protein OsWRKY108 of claim 1, characterized in that, The nucleic acid molecule is the cDNA molecule or genomic DNA molecule of SEQ ID No.

2.

3. A substance for regulating the expression of the nucleic acid molecule of claim 2, characterized in that, It is any one of the following F1, F2 or F3: F1) Targeting the sgRNA, siRNA, shRNA, miRNA, antisense RNA, or ribozyme of the nucleic acid molecule described in claim 2; F2) is a DNA molecule that encodes the sgRNA, siRNA, shRNA, miRNA, antisense RNA, or ribozyme described in F1); F3) is an expression vector containing the DNA molecule described in F2).

4. The substance according to claim 3, characterized in that, The target sequence of the sgRNA is the nucleotide sequence of positions 63-82 or 392-411 of the nucleic acid molecule SEQ ID No. 2 of claim 2.

5. The application of the protein OsWRKY108 as described in claim 1, the nucleic acid molecule as described in claim 2, or the regulatory substance as described in claim 3 in regulating the cold tolerance of rice seedlings or in cultivating cold-resistant rice varieties.

6. The application of the protein OsWRKY108 as described in claim 1, the nucleic acid molecule as described in claim 2, or the regulatory substance as described in claim 3 in the preparation of a product for regulating the cold tolerance of rice seedlings.

7. A reagent for improving the cold tolerance of rice seedlings, characterized in that, The active ingredient is the regulatory substance described in claim 3.

8. A method for improving the cold tolerance of rice seedlings, characterized in that, The method includes the following steps: inhibiting the expression of the protein OsWRKY108 as described in claim 1 in the recipient rice, reducing the abundance of the protein, and / or knocking out the encoding gene as described in claim 2, to obtain the target rice with higher cold tolerance during the seedling stage than the recipient rice.

9. The method according to claim 8, characterized in that, The inhibition, reduction, or knockout is achieved through a CRISPR / Cas9 system; the CRISPR / Cas9 system includes plasmids containing Cas9 and sgRNA.

10. A biomaterial related to the protein OsWRKY108 of claim 1, characterized in that, It is any one of B1) to B5) below: B1) A nucleic acid molecule containing the protein of claim 1; B2) An expression cassette containing the nucleic acid molecule of claim 2; B3) A recombinant vector containing the nucleic acid molecule of claim 2, or a recombinant vector containing the expression cassette of claim B1; B4) A recombinant microorganism containing the nucleic acid molecule of claim 2, or a recombinant microorganism containing the expression cassette of B1), or a recombinant microorganism containing the recombinant vector of B2); B5) A transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule of claim 2, or a transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the expression cassette of claim B2).

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