Application of WRKY35 gene in regulating cold tolerance of rice at booting stage

Abstract

This invention discloses the application of the WRKY35 gene in regulating cold tolerance during the booting stage of rice. The amino acid sequence encoded by the WRKY35 gene is shown in SEQ ID NO.3. This invention provides, for the first time, the application of the WRKY35 gene in regulating cold tolerance during the booting stage of rice. Increasing its expression level or protein activity can significantly enhance the cold tolerance of rice during the booting stage, reduce abnormal grain development caused by low-temperature stress, and thus significantly improve the seed setting rate and final yield under low-temperature conditions. This provides a novel gene resource for stable rice breeding and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, and in particular to the application of the WRKY35 gene in regulating cold tolerance during the booting stage of rice. Background Technology

[0002] Rice originated in tropical and subtropical regions and has a relatively weak ability to adapt to low-temperature environments, especially during the critical booting stage of its growth. Low temperatures significantly affect rice growth and development, leading to inhibited pollen development, reduced seed setting rate, and consequently, yield loss. Particularly in the rice-growing areas of northern my country, large-scale outbreaks of chilling injury occurring during the booting stage occur on average every 3-5 years, while smaller-scale outbreaks happen almost every year, becoming a significant limiting factor in rice production in this region. A deep understanding of the molecular mechanisms by which rice responds to low temperatures during the booting stage, the identification of key cold-resistant genes, and the subsequent use of molecular design to breed cold-resistant rice varieties are the most effective ways to prevent chilling injury.

[0003] Currently, research on cold tolerance in rice mainly focuses on the germination and seedling stages, with relatively little research on cold tolerance during the booting stage. The scarcity of cold-tolerant genes and unclear molecular mechanisms during the booting stage severely hinders the breeding process of cold-tolerant rice. Therefore, it is urgent to deeply explore the key genes controlling cold tolerance during the booting stage of rice, elucidate their regulatory molecular and physiological mechanisms, and provide effective gene resources for the molecular design of cold-tolerant rice varieties during the booting stage. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes the application of the WRKY35 gene in regulating cold tolerance during the booting stage of rice.

[0005] This invention provides the application of the WRKY35 gene in regulating cold tolerance during the booting stage of rice, and the amino acid sequence encoded by the WRKY35 gene is shown in SEQ ID NO.3.

[0006] In some implementations, the adjustment specifically includes any one of the following:

[0007] (i) Reduce cold tolerance during the booting stage by inhibiting or reducing the activity or expression level of the protein encoded by the WRKY35 gene;

[0008] (ii) Reduce cold tolerance during the booting stage by knocking out, suppressing or silencing the expression of the WRKY35 gene.

[0009] In some implementations, the adjustment specifically includes any one of the following:

[0010] (a) Improve cold tolerance during the booting stage by increasing the activity or expression level of the protein encoded by the WRKY35 gene;

[0011] (b) Improve cold tolerance during the booting stage by increasing the expression level of the WRKY35 gene.

[0012] In some embodiments, the nucleotide sequence of the WRKY35 gene is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0013] This invention also provides a method for regulating cold tolerance during the booting stage of rice by regulating the expression or activity of the WRKY35 gene or its encoded protein;

[0014] The amino acid sequence encoded by the WRKY35 gene is shown in SEQ ID NO.3.

[0015] In some implementations, the method is selected from any of the following:

[0016] (1) Downregulate the expression level of the WRKY35 gene, the expression level of the protein encoded by the WRKY35 gene, or the activity of the protein encoded by the WRKY35 gene, thereby reducing the cold tolerance of rice during the booting stage.

[0017] (2) Upregulate the expression level of the WRKY35 gene, the expression level of the protein encoded by the WRKY35 gene, or the activity of the protein encoded by the WRKY35 gene, thereby improving the cold resistance of rice during the booting stage.

[0018] In some embodiments, downregulating the expression or activity of the WRKY35 gene or its encoded protein specifically means either knocking out or silencing the WRKY35 gene in rice, or inhibiting the activity of the WRKY35 protein.

[0019] In some embodiments, the upregulation of the expression level of the WRKY35 gene, the expression level of the protein encoded by the WRKY35 gene, or the activity of the protein encoded by the WRKY35 gene in step (2) specifically refers to any one of the following:

[0020] (a) Transform the expression construct or vector of the WRKY35 gene into rice or rice cells;

[0021] (b) Transform an expression construct or vector containing the WRKY35 gene into rice or rice cells.

[0022] The present invention also provides a method for breeding rice, comprising the following steps: introducing a substance that increases or inhibits the expression level of the WRKY35 gene, the expression level of the protein encoded by the WRKY35 gene, or the activity of the protein encoded by the WRKY35 gene into rice or rice cells to obtain bred rice; wherein the amino acid sequence encoded by the WRKY35 gene is shown in SEQ ID NO.3.

[0023] The present invention also provides a method for screening regulators that regulate cold tolerance during the booting stage of rice, the method comprising: adding candidate substances to a system containing the WRKY35 gene or its encoded protein; detecting the system and observing the expression or activity of the WRKY35 gene or its encoded protein; wherein the amino acid sequence encoded by the WRKY35 gene is shown in SEQ ID NO. 3.

[0024] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0025] 1. This invention provides for the first time the application of the WRKY35 gene in regulating the cold tolerance of rice during the booting stage. It can significantly enhance the cold tolerance of rice during the booting stage and effectively reduce the abnormal grain development caused by low temperature stress, thereby significantly improving the seed setting rate and final yield. It provides a new approach for high-yield and high-quality rice breeding and has broad application prospects.

[0026] 2. This invention reduces the dependence of rice on climatic conditions by regulating the WRKY35 gene, enabling it to maintain high yield and stable quality in various environments. It provides an effective gene resource for rice cultivation in cold regions and has broad potential for industrial application. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the mutation sites of the mutant strains CR-1 and CR-2 prepared in Example 1 of the present invention;

[0029] Figure 2 This is a diagram showing the results of anther morphology and pollen grain viability detection in Example 1 of the present invention;

[0030] Figure 3 This is a graph showing the statistical results of the seed setting rate of mutant strains CR-1 and CR-2 in Example 1 of the present invention;

[0031] Figure 4 This is a graph showing the statistical results of fruit set rate after overexpression of the WRKY35 gene in Example 1 of the present invention;

[0032] Figure 5Figure 2 shows the results of WRKY35 gene mRNA expression detection in different tissues in Example 2 of this invention; Root; Stem; Leaf; Panicle; Figure a shows the qPCR detection results; Figure b shows the in situ hybridization results.

[0033] Figure 6 This is a diagram showing the subcellular localization results of WRKY35 in Example 3 of the present invention;

[0034] Figure 7 This is a graph showing the results of luciferase activity detection in Example 4 of the present invention. Detailed Implementation

[0035] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0037] Example 1: Study on the relationship between the WRKY35 gene and cold tolerance phenotype in rice during the booting stage.

[0038] Material preparation: In this embodiment, through the cold tolerance test and screening of rice during the booting stage, a cold-tolerant rice variety Longdao 5 (LD5) and a non-cold-tolerant rice variety Longjing 11 (LJ11) were obtained.

[0039] CRISPR / Cas9 editing vector design and construction: Based on the rice WRKY35 gene sequence, an appropriate sgRNA target was selected using an online design tool. The sgRNA sequence targeting the WRKY35 gene was inserted into a plant expression vector containing Cas9. The constructed CRISPR / Cas9 editing vector was then transformed into Agrobacterium strain.

[0040] The sgRNA sequence is SEQ ID NO.4.

[0041] The CRISPR / Cas9 vector is pYLCRISPR / Cas9Pubi-H.

[0042] Rice conversion:

[0043] Infection: Embryogenic callus from LD5 rice seedlings was co-cultured with Agrobacterium for 3 days to ensure that the CRISPR / Cas9 editing vector entered the rice cells.

[0044] Screening: The co-cultured callus tissue was transferred to a culture medium containing resistance markers to screen for positive plants.

[0045] Regeneration: Regenerated plants are obtained through callus differentiation culture.

[0046] Using PCR and sequencing techniques, two lines with the WRKY35 gene successfully knocked out were obtained and named CR-1 and CR-2. Figure 1 As shown.

[0047] Subsequently, rice plants of LD5, LJ11, CR-1, and CR-2 were placed in a cold chamber at 15℃ for 7 days during the booting stage to simulate the low-temperature environment of the booting stage. Anther development was observed, pollen activity was examined using an optical microscope, and changes in seed setting rate after cold treatment were detected for each line.

[0048] Pollen viability assay: Rice anthers were collected and stained with iodine-potassium iodide solution (I2-KI solution). After staining, pollen viability was observed under a microscope. Viable pollen grains appeared dark after staining, while inactive pollen grains remained transparent or light-colored.

[0049] Seed setting rate test: After low temperature treatment, the rice plants resumed normal growth until the seeds matured. The seed setting rate of all rice plants was statistically analyzed, and the seed setting rate of each plant was calculated.

[0050] Pollen activity test results are as follows Figure 2 As shown, after low-temperature treatment, the anthers of mutant strains CR-1 and CR-2 were short, whitish, and abnormally developed, with no viable pollen grains produced. The seed set rate results are as follows... Figure 3 As shown, under normal growing conditions, the seed setting rate of mutants CR-1 and CR-2 was not significantly different from that of LD5. However, after low-temperature treatment, the seed setting rate of mutants CR-1 and CR-2 differed significantly, decreasing to almost sterility. This indicates that knockout of the WRKY35 gene can reduce cold tolerance during the booting stage of rice.

[0051] WRKY35 gene CDS amplification: Total RNA was extracted from cold-tolerant rice LD5, and the full-length cDNA of the WRKY35 gene was amplified using reverse transcription PCR (RT-PCR). Specific primers were designed to clone the open reading frame (ORF) of the WRKY35 gene and insert it into a plant overexpression vector.

[0052] Upstream primer CP1133: SEQ ID NO.5;

[0053] Downstream primer CP1134: SEQ ID NO.6.

[0054] Construction of the overexpression vector XF4292: The WRKY35 gene was inserted into a plant expression vector containing a promoter and a resistance marker to construct the WRKY35 overexpression vector. The WRKY35 overexpression vector was transformed into embryogenic callus tissues of the CR-2 and LJ11 lines using Agrobacterium-mediated transformation. After co-culture, successfully transformed plants were selected using resistance screening for the detection of cold tolerance phenotypes.

[0055] Subsequently, referring to the above cold stress treatment, the changes in seed setting rate of CR-2 and LJ11 lines overexpressing WRKY35 were statistically analyzed.

[0056] The results are as follows Figure 4 As shown, overexpression of the WRKY35 gene partially restored the cold tolerance phenotype of the CR-2 and LJ11 lines, significantly improving their cold tolerance. This indicates that the WRKY35 gene plays an important role in the regulation of cold tolerance and can serve as a key target gene for improving the cold tolerance of rice.

[0057] Example 2: Tissue-specific expression analysis of the rice WRKY35 gene

[0058] Material selection: Rice seedlings, young rice panicles, leaves and other tissues were selected as materials for WRKY35 gene expression analysis.

[0059] Reagent preparation: Extract total RNA using a plant RNA extraction kit; design specific qPCR primers for the WRKY35 gene; prepare probe synthesis reagents for in situ hybridization.

[0060] Upstream primer CP4150: SEQ ID NO.7;

[0061] Downstream primer CP4151: SEQ ID NO.8.

[0062] qPCR detection: Total RNA was extracted from different tissues of rice, including young panicles, leaves, and roots. RNA was reverse transcribed into cDNA using a reverse transcription kit. The expression level of WRKY35 in different tissues was detected by qPCR using the specific primers CP4150 and CP4151 for the WRKY35 gene, and the expression differences among tissues were compared.

[0063] In situ hybridization: Based on the cDNA sequence of the WRKY35 gene, specific probes are designed and synthesized, and then labeled to facilitate subsequent detection.

[0064] WRKY35 sense probe synthesis primers:

[0065] CP1756: SEQ ID NO.9

[0066] CP1757: SEQ ID NO.10.

[0067] WRKY35 anti-sense probe synthesis primers:

[0068] CP1758: SEQ ID NO.11;

[0069] CP1759: SEQ ID NO.12.

[0070] qPCR test results as follows Figure 5 As shown in figure a, the WRKY35 gene is highly expressed mainly in young rice panicles. The results of in situ hybridization are as follows: Figure 5 As shown in b, the WRKY35 gene is highly expressed in the anther chambers of rice, suggesting that the WRKY35 gene is involved in the development of rice anthers and pollen grains.

[0071] Example 3: Subcellular localization of the WRKY35 gene

[0072] Following the method described in Example 1, the coding region of the WRKY35 gene was amplified, and transient expression vectors fused to the C and N ends of the WRKY35 gene with eGFP were constructed. After transformation into rice protoplasts, their expression and subcellular localization were observed.

[0073] 1) Vector construction: Based on the full-length sequence of WRKY35, specific primers were designed to amplify its coding region. Two eGFP fusion vectors were constructed, with eGFP sequences ligated to the C-terminus and N-terminus of the WRKY35 gene, respectively, and inserted into the plant transient expression vector.

[0074] The eGFP-WRKY35 was constructed using the vector XF2838, with upstream primer CP2193 (SEQ ID NO.13) and downstream primer CP2194 (SEQ ID NO.14).

[0075] The vector for constructing WRKY35-eGFP was cGFPXF2843, with upstream primer CP2195: SEQ ID NO.15; and downstream primer CP2196: SEQ ID NO.16.

[0076] 2) Take young rice leaves, digest the cell walls with cellulase and pectinase to separate rice protoplasts, and obtain a purified protoplast suspension by filtration and centrifugation.

[0077] 3) The constructed eGFP-WRKY35 and WRKY35-eGFP fusion vectors were introduced into rice protoplasts, respectively, and then... Transient expression transformation was performed using a mediated method. After transformation, protoplasts were cultured in the dark at 28°C for 12 hours.

[0078] 4) The fluorescence signal of the WRKY35 protein fused with eGFP at the C-terminus and N-terminus was observed in rice protoplasts using laser confocal scanning microscopy. The results are as follows: Figure 6 As shown, WRKY35 cells fused with eGFP at both the C and N ends are located in the cell nucleus.

[0079] Example 4: Detection of WRKY35 transcription factor activity

[0080] This embodiment uses a dual-luciferase reporter gene assay system to detect whether WRKY35 is an activating or repressive transcription factor. First, a recombinant WRKY35 genomic DNA sequence and coding sequence (CDS) N-terminus fused with GAL4 BD were constructed, with VP16 as a positive control and an empty GAL4 DBD vector as a negative control. Next, luciferase activity was detected after transformation into rice protoplasts. The specific steps are as follows:

[0081] 1) Experimental materials: rice protoplasts, WRKY35 genomic DNA sequence and coding sequence (CDS), GAL4 BD vector, VP16 (positive control) and GAL4 DBD empty vector (negative control).

[0082] 2) Construction of WRKY35-GAL4 BD recombinant plasmid: The genomic and coding sequences of WRKY35 were amplified, and each sequence was cloned into a vector containing GAL4 BD, ensuring that WRKY35 and GAL4 BD were fused within the same reading frame to detect its activation effect on the luciferase gene. VP16 was used as a positive control, and the empty GAL4 DBD vector was used as a negative control to ensure the accuracy and validity of the experimental results.

[0083] Among them, the upstream primer CP3490 used in the vector construction is SEQ ID NO.17;

[0084] The downstream primer CP3491 constructed from the vector: SEQ ID NO.18.

[0085] 3) Protoplast isolation and transformation: Protoplasts were extracted from rice seedlings. The cell walls were digested using cellulase and pectinase, and high-purity protoplasts were obtained by filtration and centrifugation. The constructed GAL4 BD-WRKY35 recombinant plasmid, VP16 positive control vector, and GAL4 DBD empty vector were then processed separately... The method and dual-luciferase reporter vector were co-transformed into rice protoplasts, and then cultured for 16 hours under suitable temperature and light.

[0086] 4) Luciferase Activity Assay: Transformed rice protoplasts were collected, and the luciferase activity of each group was detected using a luciferase reporter gene assay kit. The luciferase activity levels of different experimental groups were measured using a fluorescence detector, and WRKY35 was used... CDS Group, WRKY35 genomic The group was compared with the VP16 positive control group and the GAL4 DBD negative control group.

[0087] The results are as follows Figure 7 As shown, WRKY35 CDS The fluorescence activity of the group was significantly higher than that of the GAL4 DBD negative control group and close to that of the positive control, indicating that WRKY35 is an activating transcription factor.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

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Claims

1. WRKY35 The application of genes in regulating cold tolerance during the booting stage of rice, the aforementioned WRKY35 The amino acid sequence encoded by the gene is shown in SEQ ID NO.3; The adjustment specifically involves: increasing... WRKY35 Increase gene expression levels to improve cold tolerance during the booting stage.

2. The application according to claim 1, characterized in that, The WRKY35 The nucleotide sequence of the gene is shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. A method for regulating cold tolerance during the booting stage of rice, characterized in that, The method is as follows: Adjusting upwards WRKY35 Increase gene expression levels to improve cold tolerance during the booting stage of rice; The WRKY35 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

3.

4. The method according to claim 3, characterized in that, The upward adjustment WRKY35 The gene expression level is any of the following: (a) will WRKY35 Gene expression constructs or vectors are transferred into rice or rice cells; (b) containing WRKY35 Gene expression constructs or vectors are transferred into rice or rice cells.

5. A method for breeding rice with improved cold tolerance during the booting stage, characterized in that, The following steps will be included: to improve WRKY35 The substance for gene expression is introduced into rice or rice cells to obtain selected rice; WRKY35 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

3.

6. A method for screening regulators to improve cold tolerance during the booting stage of rice, characterized in that, The method includes: adding the candidate substance to a solution containing... WRKY35 In a system containing a gene or its encoded protein; detect the system and observe. WRKY35 The expression or activity of a gene or its encoded protein; WRKY35 The amino acid sequence encoded by the gene is shown in SEQ ID NO.3.

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