Use of clrlk1 gene in controlling watermelon disease resistance

Abstract

This invention discloses the application of the ClRLK1 gene in controlling watermelon disease resistance. The ClRLK1 gene cloned and verified in this invention has the DNA sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.2. The ClRLK1 gene is used to improve plant resistance to fungal diseases, specifically watermelon wilt or vine blight. This invention, through seedling resistance identification of plants overexpressing ClRLK1, demonstrates that ClRLK1 is a positive regulator of watermelon disease resistance response, confirming the function and application pathway of this gene.

Description

Technical Field

[0001] This invention relates to the field of watermelon genetic engineering. Specifically, it relates to the isolation and cloning of the watermelon ClRLK1 gene, which can improve the disease resistance of watermelons, and its application in the genetic improvement of watermelon disease resistance. Background Technology

[0002] Watermelon (Citrullus lanatus (Thunb.) Mansfeld. et Nakai) is an important horticultural crop. my country is the world's largest producer and consumer of watermelons, ranking first globally in both planting area and yield (FAO, 2021). With the increasing importance of watermelons in promoting agricultural restructuring and developing modern, efficient agriculture, watermelon cultivation is showing a year-on-year growth trend. Soil-borne fungal diseases such as Fusarium wilt and gynostemma pentaphyllum cause serious damage to watermelon production, making the breeding of disease-resistant watermelon varieties of great significance. The discovery and identification of disease-resistant genes are fundamental to breeding disease-resistant varieties, but currently, there are no reports on disease-resistant genes related to watermelons.

[0003] Receptor-like kinases (RLKs) are a class of transmembrane proteins that act as cell surface receptors to sense and transmit extracellular signals, participating in a wide range of biological functions, including regulating plant growth and development (López-Girona et al., Sci Rep 2017, 7:6714), responding to biotic and abiotic stresses (Wang et al., PLoS One 2017, 12:e0172869), mediating hormone and peptide signal transduction (Hartmann et al., Plant J 2014, 78:192-202), and plant innate immunity (Yeh et al., Plant Cell, 2016, 28:1701-1721). Studies have found that RLK proteins exhibit both functional redundancy and a degree of specificity in regulating plant innate immunity. For example, the Arabidopsis thaliana RLK proteins FLS2 and EFR recognize bacterial flagellin flg22 and elongation factor EF-Tu (or elf18 / elf26), respectively, and then bind to ligand BAK1 / SERK3 to form a ligand-inducible complex, activating Arabidopsis resistance to *Pseudomonas syringae* (Yeh et al., *Plant Cell*, 2016, 28:1701-1721). The tomato LRR-RLK gene FLS3 enhances the leaf immune response to bacterial infection by recognizing bacterial flagellin flgII-28 (Hind et al., *NatPlants*, 2016, 2:16128). The potato LRR-RLK gene StSERK3A / BAK1 can recognize the NbLRK1 protein of late blight pathogen, initiating an immune response in potatoes against the fungal disease late blight (Tian Zhejuan, 2014). The bean RLK protein PVPPGIP exhibits different modes of action against *Aspergillus niger* and *Fusarium moniliforme*. PVPGIP1 interacts with *Aspergillus niger* but not with *Fusarium moniliforme*, while PVPGIP2 interacts with both (Federici et al., *Trends in Plant Science*, 2006, 11:65-70). In summary, RLKs play an important role in plant innate immunity. However, to date, only a few RLKs have had their functions elucidated. For example, of the more than 600 receptor kinases in *Arabidopsis thaliana*, only a few dozen have had their functions revealed (Yasuda et al., *Curr Opin Plant Biol*, 2017, 38:10-18), and the types of pathogens resisted by RLKs vary among different crops. Most RLKs have not yet been identified or isolated, and their related biological functions remain to be verified. Currently, there are no reports on the application of RLK genes in improving disease resistance in watermelon. The ClRLK1 gene involved in this invention belongs to the watermelon receptor kinase family. Identifying its function in improving watermelon disease resistance is of great significance for breeding new disease-resistant watermelon varieties and for genetic improvement of plant disease resistance. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects of the existing technology, and it relates to a receptor kinase gene ClRLK1 and its application in the improvement of watermelon disease resistance.

[0005] An application of the ClRLK1 gene in improving the disease resistance of watermelon, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0006] An application of the ClRLK1 gene in improving the disease resistance of watermelon, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0007] Application of the ClRLK1 gene in improving plant resistance to fungal diseases.

[0008] As a preferred embodiment of the present invention, the fungal disease is watermelon wilt or vine blight.

[0009] The expression vector carrying the ClRLK1 gene of this invention can be introduced into plant cells using conventional biotechnological methods such as plant virus vectors, DNA transformation, and electroporation. The expression vector containing the ClRLK1 gene of this invention can be used to transform hosts, including various plants such as watermelon, for breeding new disease-resistant plant varieties. Since the gene of this invention is induced by pathogens, it can be combined with the inducible promoter of any pathogen of interest, ligated into a suitable expression vector, and transformed into a plant host. Under pathogen infection conditions, gene expression can be induced, thereby improving the plant's disease resistance.

[0010] The beneficial effects of this invention are as follows: This invention is the first to clone a watermelon receptor kinase encoding gene, ClRLK1, from the watermelon genome and verify the function of this gene. Watermelon plants overexpressing the ClRLK1 gene of this invention exhibit significantly improved disease resistance compared to wild-type controls, indicating that the ClRLK1 gene plays an important role in enhancing plant disease resistance. The ClRLK1 gene of this invention is of great significance for cultivating disease-resistant plants. Attached Figure Description

[0011] Figure 1 This is a PCR amplification diagram of the watermelon ClRLK1 gene;

[0012] Figure 2 This is a diagram showing the stress-induced expression of the ClRLK1 gene in watermelon.

[0013] Figure 3 To induce a wilt resistance phenotype in watermelon plants by overexpressing ClRLK1;

[0014] Figure 4 To establish a disease resistance phenotype for watermelon plants overexpressing ClRLK1 against anthracnose. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and specific examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0016] Explanation of the sequence list:

[0017] The sequence listing SEQ ID NO:1 is the nucleotide sequence of the ClRLK1 gene isolated and cloned in this invention, with a sequence length of 1308 bp.

[0018] The sequence listing SEQ ID NO:2 is the protein sequence encoded by the ClRLK1 gene of this invention.

[0019] The following embodiments define the present invention and describe the methods for cloning the DNA fragment of the complete coding region of the ClRLK1 gene, constructing the ClRLK1 gene overexpression vector, and verifying the function of the ClRLK1 gene. Based on the following description and these embodiments, those skilled in the art can determine the essential features of the present invention, and various changes and modifications can be made to the present invention to suit different uses and conditions without departing from the spirit and scope thereof.

[0020] Example 1: Isolation and cloning of the watermelon ClRLK1 gene

[0021] Using cDNA from root tissue of watermelon variety 'SM1' as a template, the CDS sequence of the ClRLK1 gene was amplified by PCR. The PCR reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 55℃ annealing for 10 sec, 72℃ extension for 15 sec, 35 cycles; 72℃ extension for 5 min. The amplified PCR product was ligated into the pClone007 vector (purchased from Qingke Company), positive clones were screened and confirmed by sequencing, and the ClRLK1 CDS sequence was obtained. The applicant named this clone pClone-ClRLK1 plasmid.

[0022] Example 2: Construction of ClRLK1 gene overexpression vector

[0023] The specific steps for constructing the overexpression vector are as follows: First, using the positive clone pClone-ClRLK1 plasmid obtained in Example 1 as a template, a DNA fragment containing the full length of ClRLK1 was amplified. The reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 55℃ annealing for 10 sec, 72℃ extension for 15 sec, 35 cycles; 72℃ extension for 5 min. The amplification primers were RLK-F (5'-taaaacgacggccagtaagcttATGTGGCTAAAATTCTTCACC-3') and RLK-R (5'-ccatgattacgccaagaagcttATGATGAGGATGGGGCTTTT-3'). The amplified fragment was directionally cloned into the pBI121 vector using the SoSoo homologous recombination kit (purchased from Qingke Company, catalog number TSV-S1). The vector was sequenced for confirmation, and the correctly sequenced clone was named pBI121-ClRLK1 for genetic transformation.

[0024] Example 3: Genetic transformation of the pBI121-ClRLK1 vector

[0025] The pBI121-ClRLK1 vector was introduced into the recipient watermelon variety 'SM1' using Agrobacterium-mediated transformation to obtain transformed plants, including the following steps:

[0026] (1) Explant culture: Sterile 'SM1' seeds were transferred to 1 / 2 MS medium and cultured in the dark at 25℃ to obtain sterile seedlings 3 days after germination. The cotyledons of the sterile seedlings were taken and cut into small pieces of 0.5cm×0.5cm, which are the explants.

[0027] (2) Infection: The explants were soaked in EHA105 Agrobacterium bacterial solution carrying pBI121-ClRLK1 vector for 10 min, and then removed and placed on sterile paper to absorb the Agrobacterium bacterial solution.

[0028] (3) Co-culture: The infected explants were transferred to co-culture medium and cultured in the dark at 25°C for 3 days. The composition of the co-culture medium was MS + 1.0 mg / L 6-BA + 0.1 mg / L IAA + 200 μM AS, pH 5.8;

[0029] (4) Selection culture: Explants co-cultured for 3 days were transferred to regeneration medium and cultured for 6 weeks to obtain regenerated plants. The regeneration medium consisted of MS + 1.0 mg / L 6-BA + 0.1 mg / L IAA + 400 mg / L termethin + 10 mg / L hygromycin, pH 5.8;

[0030] (5) Rooting: When the regenerated plantlets grow to 1.5cm, they are transferred to a rooting medium to root. The rooting medium consists of 1 / 2 MS + 1.0 mg / L IBA, pH 5.8.

[0031] (6) Transplanting: Take out the seedlings, wash off the culture medium on the roots, transplant them into the seedling substrate, and place them in the greenhouse for cultivation.

[0032] Example 4: Identifying the disease resistance of plants overexpressing the ClRLK1 gene

[0033] The identified ClRLK1 gene overexpression material and wild-type control were sown in 50-well trays. The experiment was repeated in triplicate. Resistance to Fusarium wilt, stem blight, and root rot was assessed separately.

[0034] Fusarium wilt resistance assessment: After the cotyledons have fully expanded, each seedling was inoculated with 5 ml of Fusarium wilt spore suspension (Fusarium wilt spore concentration was 1×10⁻⁶) using the root irrigation method. 5 The disease incidence rate was assessed and recorded 21 days after inoculation (number of bacteria / mL). Compared with the wild-type control, the wild-type control had a disease incidence rate of 83.4% 21 days after inoculation, indicating high susceptibility; the ClRLK1 gene overexpression plants had a disease incidence rate of 28.6%, indicating moderate resistance. The plants exhibited a wilt-resistant phenotype. The experimental results are as follows: Figure 3 As shown.

[0035] Anthracnose resistance assessment: When the plants reached the three-leaf stage, foliar spraying was used to inoculate against anthracnose fungus. The concentration of the anthracnose fungal spore suspension was 1×10⁻⁶. 6 The disease incidence rate was measured at 1 / mL, and the disease status of plants was investigated and recorded 5 days after inoculation. Compared with the wild-type control, 5 days after inoculation, the disease index of the wild-type control was 67.02, and the resistance evaluation was susceptible; the disease index of the ClRLK1 gene overexpression plants was 41.5, and the resistance evaluation was moderately resistant. The plants exhibited a resistant phenotype to anthracnose. The experimental results are as follows: Figure 4 As shown.

[0036] Root rot resistance assessment: When the plants reach the two-leaf-one-heart stage, they are inoculated with root rot pathogens using the root dipping inoculation method. The seedlings are immersed in a suspension of root rot pathogen spores (concentration 10). 6 The plants were soaked in a solution of ClRLK1 (number per mL) for 30 minutes, then cultured in the substrate. The disease incidence was assessed 20 days after inoculation. Compared to the wild-type control, the disease incidence of plants with overexpression of the ClRLK1 gene was comparable, suggesting that the ClRLK1 gene does not participate in the plant's immune response to root rot pathogens.

Claims

1. An overexpression ClRLK1 The application of genes in improving watermelon resistance to watermelon wilt or anthracnose is characterized by, The ClRLK1 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

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