Application of ClPR1 protein in improving the resistance of Cucurbitaceae plants to viral diseases

Abstract

The present invention discloses the application of ClPR1 protein in improving the resistance of Cucurbitaceae plants to viral diseases, and belongs to the field of genetic engineering. The present invention finds that the virus content in Cucurbitaceae plants such as melon and watermelon can be significantly reduced by overexpressing ClPR1 protein in Cucurbitaceae plants such as melon and watermelon, indicating that ClPR1 protein can improve the resistance of host plants to viral diseases. Therefore, ClPR1 protein and its encoding gene can be used for the cultivation and identification of anti-viral plant varieties required for agriculture and ecological environment management, and plays an important role in the field of plant disease resistance breeding.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to the application of ClPR1 protein in improving the resistance of cucurbitaceae plants to viral diseases. Background Art

[0002] Viral diseases are a special type of crop diseases. They spread quickly and are difficult to prevent and control. They are known as "plant cancer". The main symptoms of infected plants are mosaic, dead spots, necrosis, deformity, and yellowing. In recent years, with the changes in climate and planting patterns, viral diseases have become more frequent, seriously affecting the production of cucurbit crops including watermelon, melon, and cucumber. In the production of cucurbit crops including watermelon, melon, and cucumber, common viral diseases include zucchini yellow mosaic virus (ZYMV), cucumber green mottle mosaic virus (CGMMV), watermelon mosaic virus (WMV), and squash aphid yellows virus (CABYV). This type of disease is highly harmful, spreads quickly, is widely distributed, and has no specific pesticides.

[0003] Pathogenesis-related proteins (PRs) are a class of low molecular weight, water-soluble proteins produced by plants after infection by pathogens or induction by abiotic factors. PR proteins were first identified in tobacco infected with tobacco mosaic virus in the 1970s, and have since been reported in other plants, all of which were infected by pathogenic oomycetes, fungi, bacteria, and viruses. The PR1 protein in its family is an inherent component of plant innate immunity, mainly involved in plant systemic acquired resistance (SAR) response, and plays an important role in plant defense response, but its biochemical activity and mode of action remain unclear. It has long been used as a marker for enhancing disease resistance and is the only PR protein without functional annotation. In plants, PR1 proteins have been shown to have anti-fungal effects, such as improving wheat resistance to leaf rust; improving tomato, tobacco, and potato resistance to Phytophthora infestans; improving cotton resistance to Verticillium wilt; and participating in soybean defense response against soybean cyst nematodes. However, among a large number of research reports on PR1 protein improving host disease resistance, there are few reports on the application of this protein to improve plant viral disease resistance, and there are no reports on PR proteins with antiviral disease function in Cucurbitaceae crops. Therefore, developing PR proteins with antiviral disease function and improving the comprehensive disease resistance of Cucurbitaceae crops is of great significance for the prevention and control of Cucurbitaceae crop diseases. Summary of the invention

[0004] The purpose of the present invention is to provide an application of ClPR1 protein in improving the antiviral disease ability of Cucurbitaceae plants to solve the problems existing in the above-mentioned prior art. ClPR1 protein can significantly improve the antiviral disease ability of Cucurbitaceae plants such as watermelon and melon.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides application of ClPR1 protein in improving the resistance of Cucurbitaceae plants to viral diseases. The amino acid sequence of the ClPR1 protein is shown in SEQ ID NO:2.

[0007] The present invention also provides the use of a gene encoding the ClPR1 protein for improving the resistance of Cucurbitaceae plants to viral diseases. The nucleotide sequence of the gene encoding the ClPR1 protein is shown in SEQ ID NO:1.

[0008] The present invention also provides application of a recombinant vector containing the coding gene in improving the resistance of Cucurbitaceae plants to viral diseases.

[0009] The present invention also provides application of a host bacteria containing the recombinant vector in improving the resistance of Cucurbitaceae plants to viral diseases.

[0010] Preferably, overexpression of the ClPR1 protein reduces the virus content in Cucurbitaceae plants and improves the ability of the Cucurbitaceae plants to resist viral diseases.

[0011] Preferably, the cucurbitaceae plants include melon and watermelon.

[0012] Preferably, the virus is a virus of the genus Potyvirus.

[0013] Preferably, the virus of the genus Potyvirus includes zucchini yellow mosaic virus, but is not limited to zucchini yellow mosaic virus.

[0014] The present invention also provides a method for improving the resistance of Cucurbitaceae plants to viral diseases, by introducing a gene encoding the ClPR1 protein into the Cucurbitaceae plants, overexpressing the ClPR1 protein, and thereby improving the resistance of the Cucurbitaceae plants to viral diseases; wherein the amino acid sequence of the ClPR1 protein is as shown in SEQ ID NO: 2, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO: 1.

[0015] Preferably, the cucurbitaceae plants include melon and watermelon, and the viral disease includes a viral disease caused by zucchini yellow mosaic virus.

[0016] The present invention discloses the following technical effects:

[0017] The present invention overexpresses the watermelon disease-resistant protein ClPR1 in Cucurbitaceae plants such as melon and watermelon, and finds that the virus content in the melon and watermelon can be significantly reduced, indicating that ClPR1 has the function of improving the host's resistance to viral diseases. Therefore, the ClPR1 protein and its encoding gene can be used for the cultivation and identification of virus-resistant plant varieties required for agriculture and ecological environment management, and plays an important role in the field of plant disease-resistant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The phenotype of the plants 15 days after the ClPR1 gene was overexpressed in melon using a virus-infectious cloning vector; Mock-1 and OE-eGFP represent positive controls, and OE-ClPR1 represents overexpression of ClPR1;

[0020] Figure 2 Plant phenotype (a) and virus content (b) 15 days after overexpression of ClPR1 gene in watermelon using virus-infectious cloning vector; Mock represents positive control, OE-ClPR1 represents overexpression of ClPR1, and Heaty represents negative control;

[0021] Figure 3 ClPR1 gene was overexpressed in melon using virus-infectious cloning vector. ZYMV was inoculated 10 days later. Disease of plants occurred 25 days later. OE-ClPR1 in the figure indicates overexpressed ClPR1, and Mock-2 indicates control.

[0022] Figure 4 This is a map of the recombinant plasmid pXT1-ZYMV-ClPR1. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The present invention targets downstream genes that may be involved in plant disease resistance by performing transcriptome sequencing analysis on virus-infected disease-resistant watermelon varieties, and through gene function verification, finds that PR1 protein can significantly improve the ability of watermelons and melons to resist viral diseases. The following is a further analysis with specific examples.

[0029] Example 1 A method for overexpressing ClPR1 gene in Cucurbitaceae crops

[0030] 1. Obtaining the ClPR1 gene

[0031] Extract watermelon disease-resistant material 938-16-B (disclosed in the following document "Study on the genetic characteristics of resistance of watermelon inbred lines resistant to zucchini yellow mosaic virus. Acta Horticulturae Sinica, 2022, 49(3): 633-640.") 7 days after inoculation with virus ZYMV, extract total RNA from new leaves, reverse transcribe to synthesize the first chain of cDNA, and design primers to obtain ClPR1 gene through PCR amplification. The length is 492bp, encoding 163 amino acids, and is named ClPR1.

[0032] Amplification primers are as follows:

[0033] ClPR1-F: 5'-atgctccagtcgggcgggcccATGGAACTTCTCATGAAGC-3';

[0034] ClPR1-R: 5'-ctggagcatcacagtgtcgac TCAATATGGCCTTTGACCTA-3'.

[0035] The amplification reaction system is as shown in Table 1:

[0036] Table 1

[0037]

[0038] The amplification reaction program was: 95°C for 3 min; 95°C for 15 s, 60°C for 20 s, 72°C for 20 s, 36 cycles; 72°C for 5 min; and stored at 4°C.

[0039] The amplified PCR product was sequenced, and its nucleotide sequence (SEQ ID NO: 1) is as follows:

[0040] ATGGAACTTCTCATGAAGCTCTTCCTCTCCCTCTTTTGTGTCTTGAGCTTTGCCATGCTGCCCTCCTCTCTTGCTCAAGACTCGCCTCAAGACTTTGTCAACGCCCACAATGCGGCACGTGCCCAAGTTGGTGTCGGTCCGGTCCATTGGGATGAAAGAGTAGCAAGTTTTGCTCGACAATATGCCAACCAACGCATCAACGACTGTAAGTTGGTGCACTCAGGTGGACCGTACGGGGAGAATATT GCATGGGGTTGGCCAGACTTATCAGGCATGGCTGCAGTTAAGATGTGGGTCGATGAGAAGCAATTCTACAATCATCGCACCAATACATGTGCGGCTGGCAAGGTGTGCGGTCATTATACTCAAGTTGTGTGGAGAAATTCAGTGAGAATCGGGTGTGCTAAAGTGAGATGCACAAACAATGGTGGTACATTCATCATTTGCAACTATGACCCACGAGGCAATATCGTAGGTCAAAGGCCATATTGA.

[0041] The amino acid sequence of the protein expressed by the gene sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2:

[0042] MELLMKLFLSLFCVLSFAMLPSSLAQDSPQDFVNAHNAARAQVGVGPVHWDERVASF ARQYANQRINDCKLVHSGGPYGENIAWGWPDLSGMAAVKMWVDEKQFYNHRTNTCAAG KVCGHYTQVVWRNSVRIGCAKVRCTNNGGTFIICNYDPRGNIVGQRPY.

[0043] 2. Construction of overexpression vector and recombinant bacteria

[0044] The pXT1-ZYMV-eGFP vector (ZYMV virus infectious cloning vector, which has been disclosed in the following document "Construction of ZYMV infectious clone carrying eGFP and its infectivity. Acta Phytopathologica Sinica, 2021, 51(5):734-740") was digested with ApaⅠ and SalⅠ enzymes, and the ClPR1 gene obtained by the above PCR was subjected to homologous recombination (Vazyme) reaction with the linearized vector, and transformed into Escherichia coli DH5α competent cells (for the specific process, refer to the instruction manual of ClonExpress II One Step Cloning Kit) to obtain the recombinant plasmid pXT1-ZYMV-ClPR1 (see Figure 4 ). Take 1 μL of the recombinant plasmid and transform the competent Agrobacterium GV3101 by freeze-thaw method. After the clones grow on the plate (LB medium containing 50 mg / L kanamycin and 50 mg / L rifampicin antibiotics), pick a single clone and culture it in LB liquid medium containing kanamycin and rifampicin antibiotics for 2 hours, then perform PCR identification of the bacterial liquid. The identification primers are ClPR1-F / ClPR1-R, and the amplification system (20 μL) includes the components shown in Table 2 below:

[0045] Table 2

[0046]

[0047] PCR reaction program: 95°C for 3 min; 95°C for 15 s, 60°C for 20 s, 72°C for 20 s, 36 cycles; 72°C for 5 min; stored at 4°C.

[0048] The PCR product was detected by electrophoresis, and the transformant with the target size of 492 bp was a positive recombinant bacterium, named GV3101 / pXT1-ZYMV-ClPR1, and stored at -70°C for future use.

[0049] 3. ClPR1 gene is overexpressed in Cucurbitaceae plants

[0050] Taking melon as an example, the ClPR1 gene was overexpressed in melon using Agrobacterium tumefaciens-mediated method. The specific method is as follows:

[0051] The GV3101 / pXT1-ZYMV-ClPR1 recombinant bacteria stored at -70°C were selected and placed in 10 mL LB medium (50 mg / L rifampicin and kanamycin were added) for overnight culture. The next day, the inoculum was transferred to 20 mL LB liquid medium containing 100 μM acetosyringone at a 2% inoculum volume and cultured until the OD value was about 1. The bacteria were collected by centrifugation at 6000 g for 5 min and then washed with 10 mmol / L - 1 MgCl2, 10mmol·L -1 MES and 100 μmol·L -1 Suspend the cells in the induction buffer of acetosyringone, let it stand at room temperature for 2 hours, and then inject it into the cotyledons of melon from the back of the leaves with a 1mL syringe. The plants inoculated with pXT1-ZYMV-eGFP (OE-GFP) and pXT1-ZYMV (Mock-1) were used as positive controls, and the plants inoculated with the induction buffer were used as negative controls. After inoculation, the plants were placed at 25-28℃ and continued to be cultured.

[0052] The method for overexpressing the ClPR1 gene in watermelon is the same as above.

[0053] After the ClPR1 gene was inserted into the ZYMV virus infectious cloning vector (pXT1-ZYMV-ClPR1) and inoculated into watermelon and melon, the virus content in the plants inoculated with pXT1-ZYMV-ClPR1 was low and the symptoms were mild compared with the control vectors, i.e., the ZYMV virus infectious cloning vector without exogenous gene insertion (pXT1-ZYMV) and the ZYMV virus infectious cloning vector with eGFP gene inserted (pXT1-ZYMV-eGFP) (see Figure 1 and Figure 2 ).

[0054] 4. Analysis of resistance of melons overexpressing ClPR1 gene to ZYMV virus

[0055] After the injection of GV3101 / pXT1-ZYMV-ClPR1 recombinant bacteria, the level of ClPR1 protein in the plant body gradually increased with the propagation of the virus. The plant phenotype was observed after 15 days. The positive plants inoculated with pXT1-ZYMV showed severe mosaic and vein necrosis symptoms, while the plants inoculated with pXT1-ZYMV-ClPR1 had almost no symptoms or very mild mosaic symptoms. At this time, the plants inoculated with pXT1-ZYMV-ClPR1 were frictionally inoculated with wild-type ZYMV virus (OE-ClPR1), and the healthy plants of the negative control were also frictionally inoculated with wild-type ZYMV virus as a control (Mock-2). The plants were continued to be cultured at 25-28°C. The plant symptoms were observed after 15 days. The Mock plants showed severe stem and leaf vein necrosis and plant growth stunting, while the treated plants only showed mosaic symptoms without symptoms of vein necrosis and growth stunting (see Figure 3 ), indicating that the ClPR1 gene has the effect of improving host resistance to viral diseases. Therefore, the ClPR1 protein and its encoding gene can be used to cultivate and identify virus-resistant plant varieties required for agriculture and ecological environment management, and play an important role in the field of plant disease resistance breeding.

[0056] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of ClPR1 protein in improving the resistance of Cucurbitaceae plants to viral diseases, characterized in that: Overexpression of the ClPR1 protein reduces the virus content in Cucurbitaceae plants and improves the ability of the Cucurbitaceae plants to resist viral diseases; the amino acid sequence of the ClPR1 protein is shown in SEQ ID NO: 2, the Cucurbitaceae plants are melons and watermelons, and the viral disease is a viral disease caused by zucchini yellow mosaic virus.

2. Use of a gene encoding ClPR1 protein for improving the resistance of Cucurbitaceae plants to viral diseases, characterized in that: Overexpression of the ClPR1 protein reduces the virus content in Cucurbitaceae plants and improves the ability of the Cucurbitaceae plants to resist viral diseases; the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 1, the Cucurbitaceae plants are melons and watermelons, and the viral disease is a viral disease caused by zucchini yellow mosaic virus.

3. Use of a recombinant vector comprising the coding gene described in claim 2 in improving the resistance of Cucurbitaceae plants to viral diseases, characterized in that: Overexpression of the ClPR1 protein reduces the virus content in cucurbitaceae plants and improves the ability of the cucurbitaceae plants to resist viral diseases; the cucurbitaceae plants are melons and watermelons, and the viral disease is a viral disease caused by zucchini yellow mosaic virus.

4. Use of a host bacteria comprising the recombinant vector as claimed in claim 3 in improving the resistance of Cucurbitaceae plants to viral diseases, characterized in that: Overexpression of the ClPR1 protein reduces the virus content in cucurbitaceae plants and improves the ability of the cucurbitaceae plants to resist viral diseases; the cucurbitaceae plants are melons and watermelons, and the viral disease is a viral disease caused by zucchini yellow mosaic virus.

5. A method for improving the resistance of Cucurbitaceae plants to viral diseases, characterized in that: The ClPR1 protein is overexpressed by introducing a gene encoding the ClPR1 protein into a Cucurbitaceae plant, thereby improving the anti-viral disease ability of the Cucurbitaceae plant; wherein the amino acid sequence of the ClPR1 protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 1; the Cucurbitaceae plant is melon and watermelon, and the viral disease is a viral disease caused by zucchini yellow mosaic virus.

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