Application of oswrky7 gene in improving cold tolerance of rice

Abstract

The application of the OsWRKY7 gene in improving the cold tolerance of rice belongs to the field of biotechnology, and discloses application of a rice transcription factor OsWRKY7 in improving the cold tolerance of rice; the genomic DNA sequence of the rice transcription factor OsWRKY7 is shown as SEQ ID NO. 1. By means of CRISPR gene targeting editing technology, the OsWRKY7 gene is knocked out or its expression is down-regulated, and a rice mutant with a function loss or down-regulated expression of the gene is obtained, which can significantly improve the cold stress tolerance. Therefore, the OsWRKY7 has a good application prospect in enhancing the cold tolerance of rice and cultivating cold-tolerant rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the OsWRKY7 gene in improving the cold resistance of rice. Background Technology

[0002] Transcription factors are crucial components of plant signal transduction, participating in plant growth, development, and stress responses. WRKY transcription factors are one of the largest plant-specific transcription factor families, characterized by the presence of one or two WRKY domains. The WRKY structure consists of two parts: an N-terminal DNA-binding domain and a C-terminal zinc finger structure. While the DNA-binding domain sequence is based on WRKYGQK, some variations exist, such as WRKYGEK, WRKYGKK, WRKYGMK, WSKYGQK, WKRYGQK, WVKYGQK, and WKKYGQK. The WRKY DNA-binding domain specifically recognizes and binds to the W-box cis-elements (TTGACC / T) in the promoter of target genes, thereby regulating gene expression. The zinc finger structures are primarily of the C2H2 and C2HC types.

[0003] WRKY transcription factors are involved in plant responses to abiotic stress. Previous studies have found that overexpression of the OsWRKY71 and OsWRKY76 genes enhances cold resistance in rice, while overexpression of the OsWRKY45-1 and OsWRKY45-2 alleles weakens cold resistance. Under low-temperature stress, the OsMADS57 transcription factor directly binds to the promoter of the cold-resistance gene OsWRKY94 and promotes its expression, enhancing cold resistance. Simultaneously, it directly promotes D14 gene expression, reducing tillering. Conversely, under normal growth conditions, OsMADS57 promotes tillering by directly inhibiting the expression of OsWRKY94 and D14 genes, maintaining normal plant growth and development. This indicates that OsMADS57 inhibits or activates the expression of the cold-resistance gene OsWRKY94 and the axillary bud tillering-related gene D14 under normal and low-temperature stress conditions, respectively, regulating the balance between rice plant growth and development and low-temperature stress.

[0004] Currently, low-temperature chilling injury is one of the major factors limiting rice yield and quality, seriously affecting food security and the quality of life in my country and even globally. Revealing the mechanisms of rice cold tolerance, thereby improving its cold resistance, is one of the important tasks for rice researchers. Existing research results indicate that multiple WRKY transcription factors play important functions in biotic stress and regulate rice resistance through complex and intertwined transcriptional cascades, but their regulatory mechanisms in abiotic stress, especially in cold signal transduction, are poorly understood. Therefore, exploring the molecular mechanisms of OsWRKY7's response to cold stress and breeding cold-resistant rice varieties is of great significance for improving rice quality and yield. Summary of the Invention

[0005] The purpose of this invention is to knock out or downregulate the expression of the rice transcription factor OsWRKY7, thereby improving the cold resistance of rice and cultivating rice varieties with strong cold resistance.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] By using gene engineering techniques such as CRISPR gene-targeted editing to knock out or downregulate the expression of the OsWRKY7 gene, rice mutants with lost or downregulated OsWRKY7 gene expression can be obtained, which can significantly improve the tolerance to cold stress. Therefore, the OsWRKY7 gene can be used to breed cold-resistant rice varieties. The rice transcription factor OsWRKY7 has any of the following nucleotide sequences:

[0008] (1) The DNA sequence listed as positions 1-1737 in SEQ ID NO.1;

[0009] (2) A DNA sequence that encodes the same protein as the DNA sequence described in (1);

[0010] The beneficial effects of this invention are as follows:

[0011] The full-length cDNA of the OsWRKY7 gene in this invention was amplified from the rice variety Zhonghua 11. Under cold treatment, the transcriptional level of the OsWRKY7 gene significantly increased (…). Figure 1 The OsWRKY7 gene was knocked out in the rice variety Zhonghua 11 using gene editing technology, resulting in an OsWRKY7 gene loss-of-function mutant. Compared with Zhonghua 11, the OsWRKY7 gene mutant lines showed significantly improved cold tolerance. Figure 2 Furthermore, the OsWRKY7 gene mutant is more sensitive to the inhibitory effect of ABA during seed germination. Figure 3 The results of this invention demonstrate that knocking out the OsWRKY7 gene using CRISPR / Cas9 technology can significantly improve the cold tolerance of rice, showing promising application prospects in the breeding of cold-resistant rice varieties. Attached Figure Description

[0012] Figure 1 A schematic diagram of the expression pattern of the OsWRKY7 gene in response to abiotic stress in rice;

[0013] Figure 2 A schematic diagram illustrating the identification of cold tolerance in wild-type (WT) and mutant (wrky7) plants;

[0014] Figure 3A schematic diagram illustrating the phenotypic identification of the response of OsWRKY7 wild-type (WT) and mutant (wrky7) plants to external ABA application. Detailed Implementation

[0015] To obtain the OsWRKY7 gene and OsWRKY7 gene knockout mutant lines of this invention, please contact Wu Tao of the College of Plant Science, Jilin University, No. 5333, Xi'an Road, Lvyuan District, Changchun City, Jilin Province, 130062, China.

[0016] Example 1: Analysis of the expression pattern of the OsWRKY7 gene

[0017] Analysis of The BAR website (https: / / bar.utoronto.ca / eplant_rice / ) revealed that the expression of the OsWRKY7 gene is induced by cold stress. Figure 1 (A). To further verify the effect of cold stress on OsWRKY7 gene expression, the expression pattern of the OsWRKY7 gene under cold treatment was analyzed by quantitative real-time PCR. The results are as follows: Figure 1 As shown, cold treatment significantly induced the expression of the OsWRKY7 gene, with the expression level reaching its peak after 24 hours of cold treatment, approximately 15 times that under normal conditions. The inventors then analyzed the OsWRKY7 promoter sequence using the NewPlace database (https: / / www.dna.affrc.go.jp / PLACE / , November 13, 2023). The results showed that its promoter region contains multiple cis-acting elements related to stress response, such as W-boxes (8), MYB recognition sites (16), and ABA-responsive elements ABRE (6), etc. Figure 1 (C). These results indicate that the OsWRKY7 gene can respond to abiotic stress.

[0018] Example 2: Identification of cold tolerance in Zhonghua 11 and its mutant (wrky7) plants

[0019] To further elucidate the function of the OsWRKY7 gene under cold stress, the inventors constructed a CRISPR / Cas9-based OsWRKY7 gene loss-of-function mutant using the rice variety Zhonghua 11 as a background. This invention selected two homozygous mutant lines (w7-1 and w7-2). Compared to Zhonghua 11, w7-1 has a 36bp deletion, with 17bp deleted in an intron and 19bp deleted in an exon. w7-2 has an insertion of a single T base. Figure 2(A). Both mutation types resulted in amino acid frameshifts. Zhonghua 11, w7-1, and w7-2 were hydroponically cultured in Kimura nutrient solution until the rice seedlings reached the three-leaf stage, then transferred to a 4℃ low-temperature incubator for 3 days. After recovering to room temperature for 7 days, the survival rate was calculated and photographed. The results showed that after cold treatment, the survival rate of Zhonghua 11 was significantly lower than that of the mutant (A). Figure 2 (B and C). In addition, the inventors tested the peroxidase activity of plants of Zhonghua 11, W7-1, and W7-2 before and after cold treatment, and the results are as follows: Figure 2 As shown in Figure D, the POD enzyme activity of OsWRKY7 gene mutant plants (w7-1, w7-2) was significantly higher than that of ZH11 after cold treatment. These results indicate that knocking out the OsWRKY7 gene using CRISPR / Cas9 technology confers cold tolerance to rice.

[0020] Example 3: Negative Regulation of ABA-Mediated Seed Germination by the OsWRKY7 Gene

[0021] ABA plays a crucial role in abiotic stress tolerance. To investigate whether the OsWRKY7 gene is involved in ABA signal transduction, the inventors disinfected seeds of Zhonghua 11, w7-1, and w7-2 with a 2.5% sodium hypochlorite solution for 40 minutes, then rinsed them five times with distilled water. Twenty seeds of each of the disinfected varieties were sown on a 1 / 2 MS solid medium (containing 0, 2.5, and 5 μM ABA), respectively. Germination rates were recorded and photographed three days later, with root length exceeding the total seed length or shoot length exceeding half the seed length as the germination standard. The results showed that under 2.5 μM and 5 μM ABA treatments, the germination rate of the mutants was significantly lower than that of Zhonghua 11, indicating greater sensitivity to ABA. Figure 3 ).

Claims

1. The application of the OsWRKY7 gene in improving the cold tolerance of rice, characterized in that, The DNA sequence of the OsWRKY7 gene is shown in SEQ ID NO.1; the method to improve the cold resistance of rice is to knock out the OsWRKY7 gene using CRISPR / Cas9 gene editing technology to enhance the cold resistance of rice.

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