Application of sugarcane ScWRKY24G gene in improving bacterial blight disease resistance of plants
By cloning the ScWRKY24G gene from sugarcane and constructing an overexpression system, the problem of controlling bacterial blight in rice was solved, achieving highly efficient resistance to bacterial blight and ensuring the yield and disease resistance of rice and sugarcane.
Patent Information
- Application Number
- CN202511037211.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-25
- Publication Date
- 2025-10-28
AI Technical Summary
Existing technologies lack safe and efficient systemic fungicides, making it difficult to effectively control bacterial blight caused by Xanthomonas oryzae pv. oryzae. Furthermore, windy and rainy weather during the rice growing season increases the risk of disease spread and outbreaks, affecting food security.
The sugarcane ScWRKY24G gene was cloned from the sugarcane variety Guitang 42, and a recombinant vector overexpression system was constructed. This system was then transferred into rice and sugarcane to increase the activity and expression level of the ScWRKY24G protein, thereby enhancing the plant's resistance to bacterial blight.
Overexpression of the ScWRKY24G gene significantly improved the resistance of rice and sugarcane to bacterial blight, reduced the size of lesions, enhanced the plant's immune response, and ensured the yield and disease resistance of superior varieties.
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Figure CN120843583A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the sugarcane ScWRKY24G gene in improving plant resistance to bacterial blight. Background Technology
[0002] Diseases are key factors inhibiting crop growth and development and reducing yield. Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating bacterial disease affecting food security in my country and other major rice-growing regions globally. Therefore, identifying and elucidating the mechanisms of plant resistance genes to bacterial blight can provide important genetic resources for stable food production.
[0003] Bacterial blight is characterized by its high mutagenicity, rapid spread, and wide distribution. It invades rice plants through wounds or hydathodes, blocking the vascular bundles and causing disease. This disease causes damage within the vascular bundles, and currently, there is a lack of safe and highly effective systemic fungicides. Furthermore, damage caused by wind, rain, and flooding during the rice growing season creates favorable conditions for outbreaks of bacterial blight, making it difficult to control once it occurs. Therefore, breeding bacterial blight-resistant varieties is an economical and effective measure for controlling the disease; discovering and identifying new resistance genes and utilizing these genes to enhance rice's resistance to bacterial blight is an effective way to control it. Summary of the Invention
[0004] The purpose of this invention is to provide the application of the sugarcane ScWRKY24G gene in improving plant resistance to bacterial blight.
[0005] To achieve the above objectives, the technical solution of the present invention is as follows:
[0006] This invention cloned the ScWRKY24G gene, which regulates rice resistance to bacterial blight, from the sugarcane variety Guitang 42. The CDS sequence of the sugarcane ScWRKY24G gene is shown in SEQ ID NO.1, with a length of 1632 bp, and the amino acid sequence of its encoded protein ScWRKY24G is shown in SEQ ID NO.2.
[0007] This invention provides the use of any one of the following substances 1)-3) in at least one of the following ac:
[0008] 1) Protein ScWRKY24G;
[0009] 2) The DNA molecule encoding the protein ScWRKY24G;
[0010] 3) Recombinant vectors, expression cassettes, transgenic cell lines, or recombinant bacteria containing DNA molecules encoding the protein ScWRKY24G;
[0011] The protein ScWRKY24G is as follows (1) or (2):
[0012] (1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 of the sequence listing;
[0013] (2) A protein derived from (1) with the same function by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing.
[0014] a) Regulate plant resistance to bacterial blight;
[0015] b) Regulate the expression of genes that resist plant bacterial blight;
[0016] c) Screening, breeding, or developing plant varieties resistant to bacterial blight.
[0017] In the above applications, the DNA molecule is any one of the following: 1)-4)
[0018] 1) The coding region is the DNA molecule shown in SEQ ID NO.1 of the sequence listing;
[0019] 2) The coding region is the DNA molecule shown in positions 1-1635 of SEQ ID NO.1 in the sequence listing;
[0020] 3) DNA molecules that hybridize with the DNA sequence defined in 1) or 2) under strict conditions and encode proteins with the same function;
[0021] 4) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA sequence defined in 1) or 2) and encodes a protein having the same function.
[0022] In the above applications, regulating plant resistance to bacterial blight manifests as limiting the establishment of bacterial blight in invasive plants, increasing the size of lesions, and improving plant resistance to bacterial blight.
[0023] In the above application, regulating the expression of plant bacterial blight resistance genes refers to promoting the expression of plant resistance genes. These resistance genes include the OsICS1, OsMKK4, OsPAL1, and OsPR1 genes.
[0024] In the above application, the ScWRKY24G recombinant vector ScWRKY24G-Flag was constructed, and Agrobacterium was transferred into it by electroporation to obtain recombinant Agrobacterium. Then, the recombinant Agrobacterium was transferred into the target plant, and after several generations of propagation, homozygous transgenic plants with improved resistance to bacterial blight were obtained. The target plant was sugarcane or rice.
[0025] In addition, the present invention also provides a method for improving plant resistance to bacterial blight, the method being (1) or (2) below:
[0026] (1) By increasing the activity of ScWRKY24G protein in the target plant, plants with higher resistance to bacterial blight than those on the base plant were obtained.
[0027] (2) By promoting the expression of the ScWRKY24G gene in the target plant, plants with higher resistance to bacterial blight than those on the base plant were obtained.
[0028] The CDS sequence of the ScWRKY24G gene is shown in SEQ ID NO.1, the amino acid sequence of the ScWRKY24G protein is shown in SEQ ID NO.2, and the target plant is sugarcane or rice.
[0029] In this context, "chassis plants" refers to plants selected as foundational platforms in the field of synthetic biology. Through gene editing, metabolic pathway modification, and other operations, these plants can be used to produce various useful compounds, proteins, biomaterials, or achieve specific biological functions. In this invention, it refers to wild-type sugarcane or rice, etc.
[0030] "Target plant" generally refers to a plant selected for a specific purpose, which needs to be studied, modified, cultivated, or utilized. Products with improved performance obtained by modifying the substrate plant (through gene editing or overexpression, etc.) are called "target plants".
[0031] One implementation method for promoting the overexpression of the ScWRKY24G gene in the target plant is to overexpress the ScWRKY24G gene using the 35S promoter.
[0032] In addition, this invention also protects a breeding method, mainly a method for cultivating transgenic plants with high expression of rice bacterial blight resistance genes and enhanced resistance to bacterial blight, specifically as follows:
[0033] The content and / or activity of ScWRKY24G protein in the target plant are increased to obtain a transgenic plant, wherein the expression level of the resistance gene in the transgenic plant is greater than that in the target plant; and / or, the transgenic plant has greater resistance to bacterial blight than that in the target plant.
[0034] In the above method, increasing the content and / or activity of ScWRKY4G protein in the target plant involves introducing the DNA molecule encoding ScWRKY24G protein into the target plant.
[0035] And / or, the enhancement of the expression level and / or activity of the ScWRKY24G protein encoding gene in the target plant is achieved by introducing the DNA molecule encoding the ScWRKY24G protein into the target plant;
[0036] In the above method, the target plant is a dicotyledonous or monocotyledonous plant, including not only sugarcane and rice, but also other plants with high homology, as long as they are suitable for gene transformation operations, such as various crops, flowering plants or forestry plants.
[0037] Advantages of this invention:
[0038] This invention cloned the gene ScWRKY24G, which regulates resistance to bacterial blight in rice, from the sugarcane variety Guitang 42, and constructed plants overexpressing the ScWRKY24G gene. Experiments demonstrated that the ScWRKY24G overexpressing plants exhibited high expression levels of the resistance gene and resistance to bacterial blight, indicating that the ScWRKY24G gene has the function of regulating the expression level of the resistance gene and resistance to bacterial blight. Overexpression of this gene can improve the resistance of superior rice varieties to bacterial blight, thereby ensuring the yield of superior rice varieties and laying the foundation for improving the resistance of major rice varieties to bacterial blight and cultivating highly resistant rice varieties. Attached Figure Description
[0039] Figure 1 The results of sequence alignment between ScWRKY24G protein and homologous proteins in rice.
[0040] Figure 2 To identify the phenotype and protein level of ScWRKY24G gene overexpression promoting cell death in tobacco, the figure shows: A is the phenotype of cell death caused by ScWRKY24G overexpression in tobacco leaves, B is the statistical data of cell death caused by ScWRKY24G overexpression in tobacco leaves, and C is the protein level detection of ScWRKY24G overexpression in tobacco.
[0041] Figure 3 Phenotypic results of inoculating rice lines overexpressing the ScWRKY24G gene (ScWRKY24G-Flag-4, ScWRKY24G-Flag-11) and wild-type rice (Nip) with bacterial blight.
[0042] Figure 4Phenotypic data of rice lines overexpressing the ScWRKY24G gene (ScWRKY24G-Flag-4, ScWRKY24G-Flag-11) and wild-type rice (Nip) inoculated with bacterial blight.
[0043] Figure 5 To identify the expression level of the resistance gene against bacterial blight in rice overexpressing the ScWRKY24G gene. Detailed Implementation
[0044] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.
[0045] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.
[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.
[0047] Unless otherwise stated, the implementation of this invention will utilize conventional botanical techniques, microbiological techniques, tissue culture techniques, molecular biology techniques, chemical techniques, biochemical techniques, DNA recombination techniques, and bioinformatics techniques that are readily apparent to those skilled in the art. These techniques have been fully explained in published literature. Furthermore, the methods employed in this invention, including DNA extraction, phylogenetic tree construction, gene editing methods, gene editing vector construction, and the acquisition of gene-edited plants, except for those used in the examples below, can all be implemented using methods already disclosed in existing literature.
[0048] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” “nucleotide,” “nucleic acid molecule,” or “polynucleotide” mean, but are not limited to, isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), naturally occurring, mutant, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, and single-stranded or double-stranded structures. These nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences of non-coding regions. These terms include a gene. “Gene” or “gene sequence” is broadly used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in a genomic sequence, and / or include coding sequences in cDNA, and / or include cDNA and its regulatory sequences. In particular embodiments, such as concerning isolated nucleic acid sequences, cDNA is preferred by default.
[0049] Unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.
[0050] Example 1: Obtaining the ScWRKY24G gene
[0051] 1. Obtaining plant cDNA and analyzing its protein sequence
[0052] RNA was extracted from the leaves of the sugarcane variety Guitang 42, and cDNA was obtained by reverse transcription.
[0053] 2. PCR amplification
[0054] Using the cDNA obtained in step 1 as a template, PCR amplification was performed using F and R primers to obtain a 1632bp PCR amplification product.
[0055] The primer sequences are as follows:
[0056] Forward primer F: 5'-ATTATGCCTCTCCCGAATTCATGACCACCTCGTCCTCC-3';
[0057] Reverse primer R:
[0058] 5'-GAAGTCCAAAGCTTCTCGAGGTCAGTGTACAGCATCGACTG-3'.
[0059] 3. Obtaining the ScWRKY24G gene
[0060] The PCR amplification products were detected by 1% agarose gel electrophoresis, recovered and purified, and then sequenced.
[0061] Sequencing results showed that a 1632bp product was obtained by PCR amplification and named the ScWRKY24G gene. Its nucleotide sequence is shown in SEQ ID NO.1 in the sequence listing. The open reading frame of this gene is positions 1-1632 of sequence 1. The amino acid sequence of the ScWRKY24G protein encoded by the ScWRKY24G gene is shown in SEQ ID NO.2 in the sequence listing.
[0062] Furthermore, using ESPript 3.0, the ScWRKY24G protein sequence induced by rice smut was compared with the protein sequence encoded by a homologous gene in rice. The results showed that the sequence similarity was 80.6%. Figure 1 However, the function of the ScWRKY24G protein in regulating rice resistance to bacterial blight is unknown, demonstrating the research value of the ScWRKY24G gene in rice resistance to bacterial blight.
[0063] Example 2: Effects of ScWRKY24G overexpression on plant immune response
[0064] 1. Preparation of recombinant vectors overexpressing the ScWRKY24G gene
[0065] Using cDNA from leaves of sugarcane variety Guitang 42 as a template, PCR amplification was performed using the aforementioned forward primer F and reverse primer R to obtain a 1632bp PCR amplification product (ScWRKY24G open reading frame ORF, whose nucleotide sequence is SEQ ID NO.1, positions 1-1632).
[0066] The PCR amplification product was ligated into the p1300-Flag expression vector digested with KpnI and BstBI to obtain p1300-ScWRKY24G-Flag (ScWRKY24G-Flag).
[0067] Sequencing revealed that the recombinant vector ScWRKY24G-Flag, which overexpresses the ScWRKY24G gene, is obtained by replacing the ScWRKY24G gene shown in positions 1-1632 of SEQ ID NO.1 in the p1300-Flag vector between the KpnI and BstBI restriction sites.
[0068] 2. Transient overexpression of the ScWRKY24G gene in tobacco leaves.
[0069] The recombinant vector ScWRKY24G-Flag obtained in step 1 above was electroporated into Agrobacterium GV3101 to obtain recombinant Agrobacterium GV3101 / ScWRKY24G-Flag.
[0070] The recombinant Agrobacterium GV3101 / ScWRKY24G-Flag was transformed into tobacco leaves using a standard transformation medium. The detailed method is as follows:
[0071] Tobacco Benzovia seeds were sown in a 1:1 vermiculite:nutrient soil medium and grown in a culture room at 22℃ with a 10h light, 14h dark photoperiod. Two weeks later, tobacco seedlings were transplanted and used for transient conversion after one month of growth. GV3101 / ScWRKY24G-Flag was activated overnight using a medium containing 50 mg / L kanamycin and 50 mg / L gentamicin. The concentration of GV3101 / ScWRKY24G-Flag was adjusted to OD using injection buffer (10 mM MES pH 5.7, 10 mM MgCl2, 0.2 g / L acetylsyleugenol). 600 =0.5. Use a 2mL syringe to draw up the inoculum and inject it into the leaf from the back of the tobacco leaf, so that the tobacco leaf appears moist.
[0072] 3. Analysis of the effects of ScWRKY24G overexpression on plant immune response
[0073] Cell death is an important immune response in plants and a result of the interaction between plants and pathogens. In *Nicotiana benthamiana*, it is common practice to test whether proteins can induce cell death to determine their involvement in regulating plant resistance to pathogen infection. Rapid cell necrosis is a typical disease resistance response in plant disease resistance mechanisms, characterized by hypersensitive reactions. Figure 2 As shown, two days after tobacco injection, tobacco leaves exhibited a clear death phenotype. Figure 2 (A) ; Statistical analysis revealed that the mortality rate of leaves from *Nicotiana benthamiana* overexpressing ScWRKY24G-Flag was as high as 60% ( Figure 2 (B in the text); and the ScWRKY24G-Flag protein was detected in tobacco leaves by western blot. Figure 2 (C in the text). This suggests that overexpression of ScWRKY24G in tobacco can induce programmed cell death, potentially enhancing plant resistance.
[0074] Example 3: Analysis of rice bacterial blight resistance to overexpression of the ScWRKY24G gene
[0075] The recombinant vector ScWRKY24G-Flag obtained in step 1 above was electroporated into Agrobacterium EHA105 (Weimi Biotechnology (Hainan) Co., Ltd.) to obtain recombinant Agrobacterium EHA105 / ScWRKY24G-Flag.
[0076] Recombinant Agrobacterium EHA105 / ScWRKY24G-Flag was transformed into callus tissue (from Weimi Biotechnology (Hainan) Co., Ltd.) induced by Nipponbare rice (hereinafter referred to as wild-type rice, Nip, WT) seeds using conventional methods. The culture medium was a standard transformation medium, and the detailed method is as follows:
[0077] Mature seeds were dehulled, sterilized, and inoculated into a callus induction medium, induced in the dark for 30 days. The callus was then transferred to a subculture medium and cultured in the dark for 20 days. The callus was infected with Agrobacterium tumefaciens already activated on a medium containing 50 mg / L kanamycin, the bacterial solution was discarded, and the callus was transferred to a co-culture medium and cultured for 3 days. The callus was washed with sterile distilled water and an antibiotic solution, the surface moisture was blotted dry, and the callus was transferred to a selection medium containing 50 mg / L hygromycin for selection. The resistant callus was then transferred to a differentiation medium and cultured under light for 40 days. Differentiated seedlings were transferred to a rooting medium and cultured under light for 15 days. Multiple propagation processes yielded T3 generation homozygous rice transgenic ScWRKY24G.
[0078] Seedlings of T3 generation transgenic ScWRKY24G rice lines (ScWRKY24G-Flag-4, ScWRKY24G-Flag-11) and wild-type rice were soaked and germinated for 3 days at normal temperature (28℃) and in darkness. The germinated rice seedlings were then sown in a greenhouse and transplanted after 2 weeks of growth. At the three-leaf stage, bacterial blight inoculum was applied to the leaves by dipping scissors in the solution and cutting off the leaf tips to complete the inoculation for bacterial blight. After 3-4 weeks of inoculation and phenotypic stability, the resistance to bacterial blight in rice was observed and statistically analyzed. Phenotypic observation is as follows: Figure 3 Statistical analysis of phenotypic data, such as Figure 4 As shown, the lesion length on the leaves of T3 generation scWRKY24G rice is significantly shorter than that of the wild type, indicating resistance to bacterial blight.
[0079] Example 4: Overexpression of the ScWRKY24G gene increases the expression level of rice resistance genes.
[0080] Leaves were taken from rice lines (ScWRKY24G-Flag-4 and ScWRKY24G-Flag-11) grown in a greenhouse for one month. RNA was extracted from the leaves, reversed to cDNA, and the expression level of bacterial blight resistance genes was detected using real-time quantitative PCR (qRT-PCR). Figure 5 As shown, the expression levels of bacterial blight resistance genes OsICS1, OsMKK4, OsPAL1, and OsPR1 in transgenic rice were higher than those in wild-type rice, indicating that overexpression of ScWRKY24G increased the expression level of bacterial blight resistance genes, which is consistent with the phenotype.
[0081] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.
Claims
1. The application of the sugarcane ScWRKY24G gene in improving plant resistance to bacterial blight, characterized by, The CDS sequence of the ScWRKY24G gene is shown in SEQ ID NO.
1.
2. The application according to claim 1, characterized in that, The plant in question is sugarcane or rice.
3. The application according to claim 1, characterized in that, By constructing the ScWRKY24G recombinant vector ScWRKY24G-Flag, Agrobacterium was electroporated into it to obtain recombinant Agrobacterium. Then, the recombinant Agrobacterium was transferred into the target plant. After several generations of propagation, homozygous transgenic plants with improved resistance to bacterial blight were obtained. The target plant was sugarcane or rice.
4. The application according to claim 3, characterized in that, The Agrobacterium is Agrobacterium GV3101 or Agrobacterium EHA105.
5. The application of the recombinant vector containing the sugarcane ScWRKY24G gene as described in claim 1 in improving plant resistance to bacterial blight.
6. A method for improving plant resistance to bacterial blight, characterized in that, The method is as follows (1) or (2): (1) By increasing the activity of ScWRKY24G protein in the target plant, plants with higher resistance to bacterial blight than those on the base plant were obtained. (2) By promoting the expression of the ScWRKY24G gene in the target plant, plants with higher resistance to bacterial blight than those on the base plant were obtained. The CDS sequence of the ScWRKY24G gene is shown in SEQ ID NO.1, the amino acid sequence of the ScWRKY24G protein is shown in SEQ ID NO.2, and the target plant is sugarcane or rice.
7. The method for improving plant resistance to bacterial blight according to claim 4, characterized in that, The method to promote the expression of the ScWRKY24G gene in the target plant is to overexpress the ScWRKY24G gene using the 35S promoter.
8. The application of the ScWRKY24G gene as described in claim 1 in increasing the expression level of rice bacterial blight resistance genes, characterized in that, Overexpression of the ScWRKY24G gene can increase the expression of resistance genes for rice bacterial blight, including OsICS1, OsMKK4, OsPAL1, and OsPR1 genes.
9. A plant breeding method, characterized in that, The method involves introducing the ScWRKY24G gene into a target plant. The CDS sequence of the ScWRKY24G gene is shown in SEQ ID NO.
1. The target plant is sugarcane or rice.