CN115807025B - Application of OsXMK1 gene in regulation and control of rice bacterial leaf streak resistance - Google Patents

Abstract

The invention discloses an application of an OsXMK1 gene in regulating and controlling bacterial leaf spot resistance of rice. According to the invention, biological function research is carried out on the OsXMK1 gene, and the fact that the OsXMK1 gene is knocked out in rice is found to enable the rice to show disease resistance weakening capability on bacterial leaf streak bacteria; the over-expression of the OsXMK1 gene in rice can enable the rice to show disease resistance enhancement capability to bacterial leaf scald, namely the OsXMK1 gene positively regulates and controls the resistance of the rice to bacterial leaf scald, and the disease resistance of the rice to bacterial leaf scald can be influenced by regulating the expression level of the OsXMK1 gene.

Description

Application of OsXMK1 gene in regulation and control of rice bacterial leaf streak resistance

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to application of an S domain protein kinase gene OsXMK1 in regulating and controlling bacterial leaf spot resistance of rice.

Background

Rice is one of the most important grain crops in the world and is a staple food for more than half of the population worldwide, so that rice production is important for grain safety in China and even the world, and diseases are one of important factors affecting rice yield. Bacterial leaf streak disease of rice caused by pathogenic seeds of Xanthomonas oryzae (Xanthomonas oryzae pv. Oryzicola, xoc for short) is one of bacterial diseases seriously harming the yield of rice, generally causes 15-25% of yield reduction of rice, and can reach 40-60% in serious cases. To date, no major genes have been located in rice that correlate with bacterial leaf streak resistance. In addition, pathogenic strains are continuously mutated under the double pressures of natural selection and manual selection, and the combined use of a plurality of resistance genes to cope with pathogenic bacteria mutation has become a leading technical means in the disease-resistant breeding work of rice. Therefore, further mining of new genetic resources related to bacterial leaf streak resistance is of great importance for disease-resistant breeding applications of rice.

The S domain protein kinase is a key branch in the plant receptor-like kinase superfamily, and the rice S domain protein kinase subfamily has 147 members in total, and is reported to be involved in rice yield traits and environmental stress regulation (Chen LJ, wuriiunghan H, zhang YQ, duan KX, chen HW, li QT, lu X, he SJ, ma B, zhang WK, lin Q, chen SY, zhang JS.2013.An S-domain receptor-like kinase, osSIK2, confers abiotic stress tolerance and delays dark-induced leaf senescence in price. Plant Physiology163 (4): 1752-1765;Pan J,Li Z,Wang Q,Yang L,Yao F,Liu W.2020.An S-domain receiver-like kinase, osESG1, regulates early crown root development and drought resistance in face.plant Science 290:110318; zou, X., qin, Z., zhang, C., liu, B, liu, J., zhang, C, lin, C, li, H.and Zhao, T, 2015.Over-expression of an S-domain receiver-like kinase extracellular domain improves panicle architecture and grain yield in face.journal of experimental botany,66 (22), pp.7197-7209), the function of the remaining proteins was not studied. Up to now, no report has been made on whether the S domain protein kinase is involved in regulating the resistance of rice to bacterial leaf streaks.

Disclosure of Invention

The invention aims to overcome the defects and the shortcomings in the prior art and provides application of an S domain protein kinase gene OsXMK1 in regulating and controlling bacterial leaf spot resistance of rice.

The above object of the present invention is achieved by the following technical solutions:

the invention clones an S domain protein kinase gene OsXMK from rice in the early stage, the nucleotide sequence of the S domain protein kinase gene OsXMK is shown as SEQ ID No.1, and the nucleotide sequence shown as SEQ ID No.1 consists of 3013 deoxyribonucleotides of the rice OsXMK1 gene and upstream and downstream non-coding sequences thereof. Deoxyribonucleotides from 1 st to 253 rd in the sequence shown in SEQ ID No.1 are upstream non-coding sequences of the OsXMK1 gene; the 254 th to 365 th deoxyribonucleotide is the first exon sequence of OsXMK1 gene; deoxyribonucleotides 366 to 452 are the first intron sequence of the OsXMK1 gene; deoxyribonucleotides 453 to 566 are the second exon sequence of the OsXMK1 gene; the deoxyribonucleotide from 567 to 647 is the second intron sequence of the OsXMK1 gene; deoxyribonucleotides 648 to 793 are the third exon sequence of OsXMK1 gene; deoxyribonucleotides 794 to 856 are the third intron sequence of the OsXMK1 gene; deoxyribonucleotides 857 to 1070 are the fourth exon sequence of the OsXMK1 gene; deoxyribonucleotides 1071 to 1572 are the fourth intron sequence of the OsXMK1 gene; deoxyribonucleotides at positions 1573 to 1810 are the fifth exon sequence of the OsXMK1 gene; deoxyribonucleotides 1811 to 1936 are the fifth intron sequence of the OsXMK1 gene; deoxyribonucleotides 1937 to 2087 are the sixth exon sequence of the OsXMK1 gene; deoxyribonucleotides 2088 to 2381 are the sixth intron sequence of the OsXMK1 gene; deoxyribonucleotides 2382 to 2711 are the seventh exon sequence of the OsXMK1 gene; deoxyribonucleotides 2712 to 3013 are downstream non-coding sequences of the OsXMK1 gene. The amino acid sequence of the expressed protein is shown as SEQ ID No. 2. It will be appreciated that modifications of the nucleotide sequence of the coding gene of the invention without altering the amino acid sequence, taking into account the degeneracy of the codons and the preferences of codons of different species, are also within the scope of the invention.

According to the invention, the research on the biological function of the OsXMK1 gene proves that the knockout of the OsXMK1 gene in rice can enable the rice to show the capability of weakening disease resistance to bacterial leaf spot bacteria; the over-expression of the OsXMK1 gene in rice can enable the rice to show disease resistance enhancement capability to bacterial leaf scald, namely the OsXMK1 gene positively regulates the resistance of the rice to bacterial leaf scald, and the disease resistance of the rice to bacterial leaf scald can be influenced by regulating the expression level of the OsXMK1 gene. Has important significance on the improvement of bacterial leaf streak resistance of rice which is an important grain crop.

Therefore, the invention firstly provides the following new uses of the S domain protein kinase gene OsXMK and the coded protein thereof:

application of OsXMK1 gene shown in SEQ ID No.1 or OsXMK1 protein shown in SEQ ID No.2 in regulating and controlling bacterial leaf spot resistance of rice.

Application of OsXMK1 gene shown in SEQ ID No.1 or OsXMK1 protein shown in SEQ ID No.2 in enhancing resistance of rice to bacterial leaf streak.

Application of OsXMK1 gene shown in SEQ ID No.1 or OsXMK1 protein shown in SEQ ID No.2 in cultivating rice varieties with enhanced resistance to bacterial leaf streak bacteria.

Specifically, the method is used for enhancing the resistance of the rice to bacterial leaf streak by positively regulating an OsXMK1 gene in the rice.

Specifically, the stable inheritance of the OsXMK1 gene over-expression transgenic rice plant is obtained by constructing an OsXMK1 gene over-expression vector and transforming the rice plant.

The invention also provides application of the overexpression vector containing the OsXMK1 gene shown in SEQ ID No.1 in enhancing the resistance of rice to bacterial leaf scald disease or cultivating rice varieties with enhanced resistance to bacterial leaf scald disease.

Preferably, the overexpression vector of the OsXMK1 gene is p35MK1-p35S-OsXMK1-GFP.

Specifically, the construction process of the p35MK1-p35S-OsXMK1-GFP comprises the steps of cloning an OsXMK1 gene fragment by using an amplification primer containing BamHI and EcoRI enzyme cutting sites, respectively carrying out linearization treatment on a p35MK1-p35S-GFP vector and an OsXMK1 gene amplification product by using restriction enzymes BamHI and EcoRI, purifying and recovering the vector enzyme cutting product and the OsXMK1 gene amplification product, connecting by using T4 DNA ligase, taking a connecting product, transforming escherichia coli, screening positive clones, sequencing to obtain positive clones containing an OsXMK1 gene sequence, and finally obtaining an overexpression vector p35MK1-p35S-OsXMK1-GFP containing an OsXMK1 target gene.

Compared with the prior art, the invention has the following beneficial effects:

the research of the invention shows that the S-domain protein kinase OsXMK1 positively regulates the resistance of rice to bacterial leaf spot, is an S-domain protein kinase for positively regulating the resistance of rice to bacterial leaf spot, and can influence the disease resistance of rice to bacterial leaf spot by regulating the expression level of OsXMK1 genes. Has important significance on the improvement of bacterial leaf streak resistance of important grain crops rice; meanwhile, the OsXMK1 gene expression is regulated and controlled by jasmonic acid, and the coding protein is fixed on the rice cell membrane, so that the novel findings are helpful for revealing the molecular mechanism of the mediated disease resistance function and providing selectable genetic resources for rice disease resistance molecular design and breeding. The invention discloses the function of S domain protein kinase in regulating rice bacterial leaf spot resistance for the first time, and expands the functional range of the protein subfamily members.

Drawings

FIG. 1 is a flow chart for identifying and separating and cloning the rice disease resistance related gene OsXMK1 gene and verifying the function of the OsXMK1 gene.

FIG. 2 is a map of the genetic transformation vector p35MK1-p35S-GFP used in the present invention. RB and LB represent the right and left border of T-DNA, hpt represents hygromycin phosphotransferase gene, 35S represents 35S promoter of cauliflower mosaic virus, GFP represents green fluorescent protein gene, NOS represents terminator sequence, respectively.

FIG. 3 shows the expression pattern of OsXMK1 gene after treatment of commercial plant hormone methyl jasmonate (MeJA) (a jasmonate derivative) with qRT-PCR for detecting flower 11 in rice varieties.

FIG. 4 is an OsXMK1 subcellular localization assay. (1) is an OsXMK1 subcellular localization; (2) as a film positioning marker; (3) is bright field; (4) is a post-merge picture.

FIG. 5 shows the result of detecting the expression level of OsXMK1 gene in an overexpressed plant using qRT-PCR in the example of the present invention.

Fig. 6 is a DNA sequence mutation analysis of transgenic plants knocked out OsXMK1 gene constructed based on CRISPR-Cas9 system in an embodiment of the present invention.

FIG. 7 shows leaf disease condition and lesion length after inoculation of OsXMK1 gene knockout rice line and OsXMK1 gene overexpressing rice line with bacterial leaf streak bacteria. The length of the disease spots of the gene knockout families #1 and #2 after being inoculated with the bacterial leaf spot pathogens is obviously longer than that of the wild type plants, and the length of the disease spots of the gene overexpression families #1 and #2 after being inoculated with the bacterial leaf spot pathogens is obviously shorter than that of the wild type plants.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

FIG. 1 is a flow chart for identifying and separating and cloning the rice disease resistance related gene OsXMK1 gene and verifying the function of the OsXMK1 gene. Firstly, separating OsXMK1 genes from cDNA of flower 11 in a rice variety, and then respectively and transiently expressing the OsXMK1 genes in protoplasts of the flower 11 in the rice variety, so as to prove that the OsXMK1 proteins are positioned in cell membranes; knocking out and over-expressing the OsXMK1 gene in the flower 11 of the rice variety, and carrying out disease resistance analysis of bacterial leaf streak of the transgenic plant. The specific procedure is as described in the examples below.

Example 1 construction of overexpression and knockout vector of Rice Gene OsXMK1

1. Construction of OsXMK1 Gene overexpression vector

Flower 11 young ears of rice varieties planted in the agricultural biological gene research center of the agricultural academy of scion of Guangdong province are taken, total RNA is extracted by using a plant RNA extraction kit (Magen company), and then target gene amplification is carried out, wherein the specific experiment is as follows: cDNA was obtained by reverse transcription (reverse transcription kit from TAKARA Co.) of 1. Mu.g of RNA of high quality (OD 260/280:1.8-2.0; OD260/230. Apprxeq. 2.0). PCR amplification was performed using the cDNA as a template and KOD FX Neo (ToyoBo Co.). The reaction system is as follows: 2 XPCR buffer 25. Mu.L, 2mM dNTPs 10. Mu.L, F primer (5'-GACTTGACGCCGGATCCATGTACG ACGTGGCCGGGGAGC-3', SEQ ID No. 3) 0.5. Mu.L, R primer (5'-CTTGACGCCGAAT TCTCGTGGCTCGATGACGGTGATGGAG-3', SEQ ID No. 4) 0.5. Mu.L, cDNA template 1. Mu.L, KOD FX Neo (1U/. Mu.L) 1. Mu.L, and water to make up to 50. Mu.L. The reaction conditions are as follows: 98 ℃ for 5min;98℃30sec,60℃30sec,68℃30sec,35 cycles; and at 68℃for 10min. After the reaction, the PCR product was collected.

The p35MK1-p35S-GFP vector (35S promoter and GFP tag containing cauliflower mosaic virus, vector map shown in FIG. 2) was linearized with restriction enzymes BamHI, ecoRI (Thermo Co.) in the following manner: 3. Mu.g of vector, 3. Mu.L of BamHI and EcoRI endonucleases, 6. Mu.L of 10 XBuffer, and water was added to 60. Mu.L. The enzyme digestion conditions are as follows: 37℃for 1 hour. After the vector cleavage product and the PCR product were purified and recovered, ligation was performed using T4 DNA ligase (Takara Co.) in the following manner: the target fragment: 1.5. Mu.L, vector fragment: 4.5. Mu.L, 5 Xbuffer 2. Mu.L, T4 DNAligenase 1. Mu.L, and water was added to make up to 10. Mu.L. The connection conditions are as follows: 25℃for 30min. All the products were taken and added to 100. Mu.L of E.coli DH 5. Alpha. Competence, and the transformed products were spread on LB solid medium (containing kanamycin resistance, kanamycin concentration was 50 mg/L). After overnight incubation at 37℃10 single clones were picked for colony PCR identification. And 2 positive clones are selected for sequencing to obtain positive clones containing the OsXMK1 gene sequence, and finally the overexpression vector p35MK1-p35S-OsXMK1-GFP containing the OsXMK1 target gene is obtained.

2. Construction of OsXMK1 gene knockout expression vector

Targets and primers for Crispr were designed via the website (http:// skl. Scau. Edu. Cn /) based on the gene sequence of OsXMK 1:

designing a CRISPR target sequence at the 2 nd exon of the OsXMK1 gene, wherein the specific target+PAM sequence is as follows: CAAGGACGCGATGAAGATCA +TGG. Corresponding primers are designed according to the sequence and used for constructing a CRISPR vector of the OsXMK1 gene, and the specific primer sequences are shown below.

ENTRY-F：GACACCTGCAGTCTAGAGGATC(SEQ ID No.5)

ENTRY-R：GCGCCATGCATACTAGTAGATC(SEQ ID No.6)

XMK1-gRNA-R：TGATCTTCATCGCGTCCTTCAACACAAGCGGCAGC(SEQ ID No.7)

XMK1-gRNA-F：GAAGGACGCGATGAAGATCAGTTTTAGAGCTAGAAATA(SEQ ID No.8)

First, obtaining OsU promoter-driven OsXMK1 gRNA scaffold sequence by using an overlay PCR method, and respectively amplifying the segment fragments by using primer pairs ENTRY-F/XMK1-gRNA-R and ENTRY-R/XMK1-gRNA-F, wherein the corresponding PCR procedures are as follows: 98 ℃ for 2min; 15s at 98 ℃, and 5 cycles of touchdown at 68-63 ℃ by a two-step method; the two-step process at 65℃was repeated for 30 cycles. Then using the 2 product fragments as templates, using Entry-F and Entry-R as primers, and setting the PCR program at 98 ℃ for 2min; a two-step process was performed for 32 cycles at 98℃for 15s and 65℃to obtain OsU master-OsXMK 1 gRNA scaffold. The product fragments obtained from the above experiments were subjected to a PstI and SpeI double cleavage reaction and ligated to the CRISPR-Cas9 binary vector using T4 DNA ligase.

Example 2 expression pattern of OsXMK1 Gene after treatment of Rice with the plant hormone methyl jasmonate

Jasmonic acid and its derivatives have been shown to be involved in plant-pathogen interactions and have important regulatory functions in the natural immune processes of plants such as rice. The embodiment detects the expression mode of the OsXMK1 gene after the rice is treated by methyl jasmonate, and the specific method comprises the following steps:

(1) Immersing wild rice seed No.11 in water at 32 ℃ for 3 days, and growing germinated rice seeds in soil for 7 days;

(2) Methyl jasmonate (Sigma-Aldrich 392707) is diluted to 1 mu M in deionized water, uniformly sprayed on the surfaces of seedlings by a spray can, and sprayed with water by a control group;

(3) Sampling at 6 time points of 0 hour, 0.5 hour, 2 hours, 6 hours, 12 hours and 24 hours after treatment, and extracting RNA for subsequent detection;

(4) qPCR detection primer: the F5'-CCACCATGTCCAACGTCCTCCTCGC-3', R5'-TCGTGGCTCGATGACGGTGATGGAG-3', PCR reaction procedure is: 95 ℃ for 2min; the cycle was 95℃for 30s,60℃for 30s, 40.

As shown in FIG. 3, compared with the control (Mock) treatment, after MeJA treatment for 6h and 12h, the expression level of the OsXMK1 gene is obviously up-regulated, and after 24h, the expression level of the OsXMK1 gene is recovered, which indicates that the expression level of the OsXMK1 gene is regulated by jasmonic acid signals.

Example 3 subcellular localization analysis of OsXMK1 protein

Subcellular localization analysis of OsXMK1 protein can be carried out by using the OsXMK1 gene overexpression vector in example 1, p35MK1-p35S-OsXMK1-GFP is transiently expressed in rice protoplast, rice seedlings which are aseptically cultured for 10-15 days are taken, leaf sheaths are rapidly cut into small sections below 1mM by using a blade, enzymolysis buffer (0.6M mannitol, 10mM MES, cellulase (1.5%), pectinase (0.75%), 0.15 bovine serum albumin, 1mM calcium chloride) is used for enzymolysis, and light-shielding reaction is carried out for 4-6 hours. Protoplast cells were then collected with W5 buffer (154 mM sodium chloride, 125m calcium chloride, 5mM potassium chloride, 2mM MES (pH 5.7)). The protoplast cells were resuspended in MMG buffer (0.6M mannitol, 15mM magnesium chloride, 4mM MES (pH 5.7)), added with p35MK1-p35S-OsXMK1-GFP vector and PEG4000 solution, immediately gently mixed upside down, and left at room temperature in the dark for 15 minutes. The transformation was then terminated by the addition of W5 buffer. The supernatant was centrifuged off, and the mixture was added with a W5 buffer solution and dark-cultured at 28℃for 12 hours.

Fluorescence was observed with confocal fluorescence microscopy, and the results are shown in fig. 4, which show that the OsXMK1 protein localizes in the cell membrane, indicating that the OsXMK1 protein is a cell membrane localization protein.

Example 4 obtaining and identification of transgenic seedlings overexpressing OsXMK1 Gene and knocked out OsXMK1 Gene

The vector for over-expressing the target gene OsXMK1 constructed in example 1 and the vector for knocking out the target gene OsXMK1 are respectively transferred into flowers 11 of japonica rice varieties by adopting an agrobacterium EHA105 mediated genetic transformation method. And obtaining the T0 generation transgenic seedling through selective culture, differentiation, rooting and seedling hardening.

1. Acquisition and characterization of transgenic seedlings overexpressing the OsXMK1 Gene

And (3) carrying out PCR identification on all transgenic seedlings, and selecting 20 positive transgenic seedlings for breeding to obtain T1 generation and T2 generation. The T2 generation seeds are germinated by using a culture medium containing hygromycin, and if the seeds can grow normally, the strain is proved to be a homozygous strain. 2 homozygous transgenic lines (OX- #1, OX- # 2) were selected for qRT-RCR detection and subjected to subsequent experiments and analysis.

The over-expression effect process of the qRT-RCR identification target gene OsXMK1 in the transgenic plant is as follows:

(1) The total RNA of four-leaf stage rice leaves is extracted by a plant RNA extraction kit (Magen company), and 1 mug of high-quality (OD 260/280:1.8-2.0; OD260/230: 2.0) RNA is taken for reverse transcription (a reverse transcription kit of TAKARA company) to obtain cDNA.

(2) The cDNA in the step 1 is used as a template, the expression condition of the OsXMK1 gene is detected through qRT-OsXMK1-F (CCACCATGTCCAACGTCCTCCTCGC, SEQ ID No. 9) and qRT-OsXMK1-R (TCGTGGCTCGATGACGGTGATGGAG, SEQ ID No. 10) primer pairs, the expression of the rice Actin2 gene is detected by adopting OsActin2-qF (TCTTACGGAGGCTCCACTTAAC, SEQ ID No. 11) and OsActin2-qR (TCCACTAGCATAGAGGGAAAGC, SEQ ID No. 12) primer pairs of rice housekeeping gene Actin2 gene as internal references, the quantitative PCR reagent is Premix ExTaqTM (TAKARA Co.), and the quantitative PCR instrument is CFX 96 (Bio RAD Co.). The reaction system is as follows: 2 XPCRbuffer 5. Mu.L, qRT-OsXMK1-F primer 0.4. Mu.L, qRT-OsXMK1-R primer 0.4. Mu.L, cDNA template 1. Mu.L, sterilized water 3.2. Mu.L, and total volume of the reaction system 10. Mu.L. The reaction procedure: 95 ℃ for 30s;95℃for 5sec,68℃for 30sec,45 cycles.

As a result, as shown in FIG. 5, the expression level of the OsXMK1 gene was greatly increased in each of the selected 2 strains (OX- #1 and OX- # 2), and these 2 overexpressed strains were subsequently selected for the experiment.

2. Acquisition and identification of transgenic seedlings with knocked-out OsXMK1 Gene

PCR amplification (Crispr-OsXMK 1-F: TGGCAAGCGAGAGATACTAG, SEQ ID No.13; crispr-OsXMK1-R: TTGGAGAAGCTGTCGGTGG, SEQ ID No. 14) is performed on the knocked-out transgenic material obtained by the OsXMK 1-specific primer pair, and the PCR product is sequenced and compared with wild genomic DNA to finally obtain the edited mutant transgenic seedling. And (5) propagating the transgenic seedlings to finally obtain the homozygous mutant transgenic material. 2 homozygous transgenic lines were selected for phenotyping.

The results of target sequencing of Crispr knockout plants are shown in FIG. 6, in which ZH11 represents the control genomic (Zhonghua 11) DNA sequence. KO- #1 and KO- #2 are homozygous knockout plants. The black underlined indicates PAM sites and the "-" on the base indicates the deleted sequence.

EXAMPLE 5 phenotypic analysis of resistance of genetically transformed OsXMK1 plants to bacterial Pyricularia

And (3) performing bacterial leaf spot pathogen inoculation test on the over-expressed and gene knockout transgenic plant and the wild control plant by using an Xoc GD method, analyzing disease resistance phenotype and counting the length of the leaf spot 14 days after inoculation. As a result, as shown in FIG. 7, the length of the lesions after the gene knockout families #1, #2 (KO- #1, KO- # 2) were inoculated with bacterial leaf spot pathogens was significantly longer than that of the wild-type plant (ZH 11), and the length of the lesions after the gene overexpression families #1, #2 (OX- #1, OX- # 2) were inoculated with bacterial leaf spot pathogens was significantly shorter than that of the wild-type plant (ZH 11). The result shows that the rice strain after the overexpression of the OsXMK1 gene can enhance the disease resistance of rice to bacterial leaf scald bacteria.

In conclusion, the rice S domain protein kinase OsXMK1 is positioned on cell membranes, so that the resistance of rice to bacterial leaf scald disease can be positively regulated, the disease resistance of rice to bacterial leaf scald disease can be weakened by knocking out the OsXMK1 gene, and the disease resistance of rice to bacterial leaf scald disease can be enhanced by over-expressing the OsXMK1 gene; can influence the disease resistance of rice to bacterial leaf streak by regulating the expression level of OsXMK1 genes.

Claims (4)

1. Comprises the sequence shown in SEQ ID No.1OsXMK1Use of a gene over-expression vector for increasing resistance of rice to bacterial leaf streak, characterized in that the use is by constructionOsXMK1The over-expression vector of the gene is converted into rice plants to obtain stable inheritanceOsXMK1The gene over-expresses the transgenic rice plant, thereby enhancing the resistance of the rice to bacterial leaf streak.

2. Comprises the sequence shown in SEQ ID No.1OsXMK1The application of the over-expression vector of the gene in cultivating rice varieties with enhanced resistance to bacterial leaf scald bacteria is characterized in that the application is by constructingOsXMK1The over-expression vector of the gene is converted into rice plants to obtain stable inheritanceOsXMK1The transgenic rice plants are subjected to gene overexpression, so that the rice varieties with enhanced bacterial leaf spot pathogen resistance are obtained.

3. The use according to claim 1 or 2, characterized in that theOsXMK1The over-expression vector of the gene is p35MK1-p35S-OsXMK1-GFP.

4. The use according to claim 3, wherein the construction of the p35MK1-p35S-OsXMK1-GFP is carried out by cloning with the use of an amplification primer containing BamHI and EcoRI cleavage sitesOsXMK1The gene fragment was then used to make a vector of p35MK1-p35S-GFP and the restriction enzymes BamHI and EcoRI, respectivelyOsXMK1Linearizing the amplified gene product, enzyme cutting the carrier, andOsXMK1purifying and recovering the gene amplification product, connecting by using T4 DNA ligase, taking the connecting product, transforming the escherichia coli, screening positive clones and sequencing to obtain the DNA-containing recombinant DNAOsXMK1Positive cloning of the Gene sequence, finally obtaining a Gene sequence containingOsXMK1The overexpression vector of the target gene p35MK1-p35S-OsXMK1-GFP.