Rice OsJacLK1 gene and application thereof in resisting rice blast

Abstract

The invention provides a rice OsJacLK1 gene and application of the rice OsJacLK1 gene in resisting rice blast. The length of a coding sequence of the rice OsJacLK1 gene is 1794 bp, and the rice OsJacLK1 gene is used for coding a Jacalin-like lectin receptor-like kinase and consists of 597 amino acids. According to the invention, an overexpression vector of the rice OsJacLK1 gene is constructed, and an OsJacLK1 gene overexpression rice plant is created by means of an agrobacterium-mediated rice mature embryo transformation technology. A magnaporthe oryzae inoculation experiment is carried out on wild type and OsJacLK1 overexpressed rice plants, and the result shows that the overexpressed OsJacLK1 gene improves the resistance of the rice plants to magnaporthe oryzae, so that the rice OsJacLK1 gene can be used for cultivating rice blast resistant rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of rice breeding, and particularly relates to a rice OsJacLK1 gene and an application thereof in rice blast resistance. Background Art

[0002] Rice (Oryza sativa L.), an annual aquatic herb native to tropical and subtropical regions, is China's most important food source and the crop with the largest cultivated area, highest yield per unit area, and highest total output. Rice production also impacts global economic development. Because rice is grown in diverse agroclimatic environments, it suffers from two main types of damage: abiotic, such as high and low temperatures, drought, and flooding; and biotic, primarily small insects (such as aphids), plant-borne bacteria, viruses, and fungi. Currently, rice blast is a major limiting factor in rice production. A serious global fungal disease of rice, rice blast causes lesions on leaves, stems, pedicels, panicles, seeds, and even roots. Globally, rice blast causes annual yield losses of 11% to 30%, threatening global food security. According to reports, under favorable conditions, the disease can destroy an entire rice plant within 15 to 20 days, resulting in yield losses of up to 100%. Among the many methods for preventing and controlling rice blast, the most environmentally friendly and cost-effective measure is to research and develop disease-resistant varieties. Therefore, cultivating new rice varieties with resistance has become an important way to achieve sustainable agricultural development.

[0003] Lectins are a class of plant proteins that contain at least one non-catalytic domain and can reversibly bind to carbohydrate structures. Jacalin-related lectins (JRLs) are proteins with one or more Jacalin domains and represent a new family within the lectin superfamily. JRL proteins play a crucial role in plant responses to biotic stresses (such as pathogens and insect pests) and abiotic stresses (such as salt stress). By binding carbohydrate ligands, lectins participate in the perception of environmental signals and their translation into phenotypic responses. In addition to regulating growth and development, JRLs can respond to a variety of biotic (such as pathogens and insect pests) and abiotic (such as drought and salinity) stresses, enhancing plant stress tolerance by regulating gene expression and protein synthesis. Furthermore, rice JRL proteins contain a Pkinase domain, the catalytic domain of protein kinases. Protein kinases catalyze protein phosphorylation, activating or inactivating them, and participate in the regulation of various cellular functions, such as cell growth, division, differentiation, cell-cell interactions, apoptosis, and environmental responses.

[0004] Lectin receptor-like kinases (LecRLKs) are a subfamily of the receptor-like protein kinase family and mainly contain three domains: an extracellular lectin domain, a transmembrane domain, and an intracellular kinase domain. In plants, LecRLKs can participate in biotic stress responses, abiotic stress responses, and plant growth. Jacalin lectin receptor protein kinases are specifically present in monocotyledons. Summary of the Invention

[0005] The present invention aims to provide a rice OsJacLK1 gene and its application in rice blast resistance. By overexpressing the rice OsJacLK1 gene for crop genetic improvement, transgenic rice resistant to rice blast can be cultivated.

[0006] In order to achieve the above objectives, the following technical solutions are adopted: The first aspect of the present invention provides a rice OsJacLK1 gene, the nucleotide sequence of which is shown in SEQ ID No.1.

[0007] The second aspect of the present invention provides a protein encoded by the rice OsJacLK1 gene, the amino acid sequence of which is shown in SEQ ID No.2.

[0008] The third aspect of the present invention provides a recombinant expression vector containing the rice OsJacLK1 gene.

[0009] The fourth aspect of the present invention provides the use of the above-mentioned rice OsJacLK1 gene in improving the resistance of rice to rice blast; Furthermore, the application is specifically: overexpressing the rice OsJacLK1 gene in rice by genetic engineering means to increase the expression level of the rice OsJacLK1 gene, thereby improving the resistance of rice to rice blast.

[0010] The fifth aspect of the present invention provides the use of the above-mentioned rice OsJacLK1 gene in breeding rice varieties resistant to rice blast; Furthermore, the application is specifically: overexpressing the rice OsJacLK1 gene in rice by genetic engineering means, thereby increasing the expression level of the rice OsJacLK1 gene and obtaining a rice variety resistant to rice blast.

[0011] The present invention uses a cold-shock method to transform an overexpression vector of the rice OsJacLK1 gene into Agrobacterium tumefaciens EHA105 competent cells. Using Agrobacterium-mediated transformation of mature rice embryos, OsJacLK1-overexpressing rice plants were obtained and spray-inoculated for evaluation of resistance to rice blast. Following infection with the blast fungus, the expression levels of genes involved in the OsP BZ1, OsPAL1, and OsAOS2 signaling pathways in the OsJacLK1-overexpressing plants were analyzed. The results indicate that overexpression of the rice OsJacLK1 gene significantly enhances rice resistance to rice blast. The rice OsJacLK1 gene may enhance plant resistance to pathogens by regulating the expression of disease-resistance-related genes.

[0012] The present invention has the following beneficial effects: Currently, there are no reports on the use of the rice OsJacLK1 gene for rice blast resistance. The present invention uses biotechnology to alter the expression level of the rice OsJacLK1 gene. Rice plants overexpressing the OsJacLK1 gene exhibit significantly improved resistance to rice blast compared to wild-type rice. Therefore, crop genetic improvement through overexpression of the rice OsJacLK1 gene can be used to cultivate blast-resistant rice varieties, demonstrating significant practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Diagram of the strategy for overexpression vector construction.

[0014] Figure 2 : Detection of the hygromycin phosphotransferase gene (HPT) fragment in the genomic DNA of the tested transgenic rice. Wells 1-11 are the tested transgenic rice seedlings, + is the pCuG-OsJacLK1 positive plasmid, M is the DNA marker, and - is the water control.

[0015] Figure 3 : qRT-PCR analysis of OsJacLK1 expression in rice plants overexpressing OsJacLK1. ZH11 is the wild-type rice Zhonghua 11, and the others are OsJacLK1-overexpressing rice plants to be tested.

[0016] Figure 4 :Incidence of rice blast in leaves of OsJacLK1-overexpressing rice.

[0017] Figure 5 : Blast lesion level in OsJacLK1 overexpressing rice.

[0018] Figure 6 : Expression of defense-related genes in OsJacLK1-overexpressing rice. DETAILED DESCRIPTION

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited thereto.

[0020] Example 1: An overexpression vector for rice OsJacLK1 (Os11g39530) was constructed and then transferred into mature rice embryos via Agrobacterium-mediated genetic transformation. The transformed rice was grown using the wild-type rice Zhonghua 11 (ZH11) as the background. OsJacLK1-overexpressing lines were screened from the progeny and their resistance to rice blast was assessed by inoculation with the rice blast fungus. The specific steps involved were: (1) Obtaining the OsJacLK1 gene and constructing its overexpression vector pCuG-OsJacLK1 ( Figure 1 The coding sequence of the OsJacLK1 gene (SEQ ID NOs. 1-2) was obtained from the rice genome database. Primers were designed using the full-length coding sequence of the OsJacLK1 gene as a template. The forward primer pCuG-OsJacLK1-F: 5'-ATGGCGGACGAGTTACAACACAC-3' and the reverse primer pCuG-OsJacLK1-R: 5'-GGATCCATGGTGAGCAAGGGCGAGGAG-3' were obtained. The OsJacLK1 gene was amplified by PCR using cDNA from rice Zhonghua 11 using PhantaMa high-fidelity enzyme, and the agarose gel was excised and recovered. The plant overexpression binary vector pCXUN-GFP (pCuG) was linearized using the restriction endonuclease SmaI, and the digestion product was recovered. Using seamless cloning technology, the amplified product of the OsJacLK1 gene was ligated in vitro with the linearized pCuG vector to obtain the ligation product, which was transformed into Escherichia coli competent cells and spread on LB plates (containing 50 mg / L Kan) for culture. The cells were inverted and cultured at 37°C for 14-16 hours to form single colonies. Positive clones were screened by colony PCR, and the positive clones with correct sequencing were taken for liquid expansion culture. The culture was mixed with glycerol at a final concentration of 15% and stored in a -80°C refrigerator.

[0021] (2) Obtaining the Agrobacterium strain of pCuG-OsJacLK1. Specific steps: Use the cold shock method to transform the positive clone plasmid of the overexpression vector pCuG-OsJacLK1 into Agrobacterium tumefaciens EHA105 competent cells, spread on LB plates (containing 50 mg / L Kan and 50 mg / L Rif), culture, and invert at 28°C for 2 days to form single colonies. Use colony PCR to screen out Agrobacterium clones carrying the target gene.

[0022] (3) Obtaining OsJacLK1 overexpression lines by infecting rice and verifying the results: Agrobacterium containing the pCuG-OsJacLK1 overexpression vector was transferred into mature embryonic callus of rice Zhonghua 11 (ZH11). Positive OsJacLK1 overexpression rice plants were obtained through screening, differentiation, and rooting. The authenticity of the plants was identified by PCR amplification of the hygromycin resistance gene (HPH) fragment. Figure 2 As shown. Leaf RNA was extracted from OsJacLK1 overexpressing plants carrying the damp mildew resistance tag, reverse transcribed into cDNA, and the basal expression of the OsJacLK1 gene in rice was detected. The results are shown in Figure 3 The results showed that compared with the wild-type rice Zhonghua 11, the OsJacLK1 gene in the OsJacLK1 overexpressing rice plants was up-regulated, with the expression level increasing by 10-100 times, indicating that the rice OsJacLK1 gene was over-expressed. Figure 3 shown.

[0023] (4) Disease resistance determination of overexpression plants: Wild-type rice Zhonghua 11 and transgenic rice overexpressing OsJacLK1 were cultured to the three-leaf, one-heart stage and spray-inoculated with spores of the nucleophilic strain Guy11 of the rice blast fungus at a concentration of 2×10 5 / mL, then, after being cultured in the dark at 23℃ overnight, the cells were transferred to the light and cultured. After 7 days, the disease level was investigated and the expression levels of the defense-related genes OsPBZ1 (LOC_Os12g36880), OsPAL1 (LOC_Os02g41630) and OsAOS2 (LOC_Os03g12500) in the cells of the plants were analyzed. Figure 4 、 Figure 5 As shown in the figure, the lesions on the leaves of wild-type rice variety Zhonghua 11 were grade 5, most of which were connected and the leaf tissue was severely necrotic; while the OsJacLK1 overexpressing rice plants showed a disease-resistant phenotype, with most of their lesions graded at 1-3, and no severe necrotic lesions were observed. This indicates that overexpressing the OsJacLK1 gene can improve rice's resistance to rice blast. Figure 6 As shown in the data, after inoculation with rice blast fungus, the expression levels of three genes, OsPBZ1, OsPAL1 and OsAOS2, in OsJacLK1-overexpressing rice plants were higher than those in wild-type rice Zhonghua 11, indicating that after being invaded by pathogens, rice can quickly induce the synthesis of jasmonic acid and further stimulate disease resistance defense responses. The rice OsJacLK1 gene can regulate the expression of disease-resistance-related genes in plants, thereby improving the plant's disease resistance response to pathogens.

[0024] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Rice OsJacLK1 A gene characterized by: The nucleotide sequence is shown in SEQ ID NO.

1.

2. The rice according to claim 1 OsJacLK1 A gene encoding a protein characterized by: The amino acid sequence is shown in SEQ ID NO.

2.

3. Rice containing the rice according to claim 1 OsJacLK1 Recombinant gene expression vector.

4. The rice according to claim 1 OsJacLK1 Application of genes in improving rice resistance to rice blast.

5. The use according to claim 4, characterized in that: Overexpression of rice in rice through genetic engineering OsJacLK1 Genes to improve rice OsJacLK1 The expression level of the gene can be increased to improve the resistance of rice to rice blast.

6. The rice according to claim 1 OsJacLK1 Application of genes in breeding blast-resistant rice varieties.

7. The use according to claim 6, characterized in that: Overexpression of rice in rice through genetic engineering OsJacLK1 Genes to improve rice OsJacLK1 The expression level of the gene was determined to obtain rice varieties with resistance to rice blast.

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