Cucumber infectious nucleic acid derived from watermelon silver mottle virus as well as construction method and application of cucumber infectious nucleic acid

Abstract

The invention discloses cucumber infectious nucleic acid derived from watermelon silver mottle virus as well as a construction method and application of the cucumber infectious nucleic acid. For the watermelon silver mottle virus epidemic in the Stone family district, segmented primers are adopted to amplify and integrate watermelon silver mottle virus genomes L RNA, M RNA and S RNA respectively, infectious full-length cDNA clone is constructed and applied to screening of cucumber resistance germplasm resources, and compared with the prior art, the method is easy to operate, high in inoculation morbidity and high in identification accuracy, and has a good application prospect. The cucumber germplasm resources can be subjected to rapid and accurate disease resistance identification in the seedling stage, the breeding efficiency is improved, the breeding cost is saved, and the breeding process is accelerated. The identification method can be applied to disease resistance identification of cucumber germplasm resources, various parents, hybrids and other materials.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a cucumber infectious nucleic acid derived from watermelon silver mottle virus and a construction method and application thereof. Background Art

[0002] Cucumber (Cucumissativus L) is one of the important vegetable crops in my country. However, in recent years, cucumber diseases have become increasingly serious, becoming an important factor restricting cucumber production, seriously affecting cucumber yield and quality.

[0003] Watermelon silver mottle virus disease is caused by watermelon silver mottle virus, which belongs to the genus Orthotospovirus of the family Tospoviridae. Watermelon silver mottle virus disease is difficult to control in production, and no specific drug has been developed to prevent and control plant viral diseases. Currently, the two main methods of control are transplanting healthy seedlings and spraying pesticides. However, pesticides can prevent and control thrips but cannot effectively prevent the virus from infecting. Moreover, the use of chemical agents for control will pollute the environment and have high costs. Therefore, early targeted prevention and control of watermelon silver mottle virus is particularly important for breeding anti-virus varieties. Summary of the invention

[0004] The present invention is based on the suspected virus infection symptoms such as mottled and deformed leaves of some cucumber plants found in Shijiazhuang City, Hebei Province. In order to further clarify the virus types that cause cucumber-related symptoms, the inventors identified the virus types of the collected cucumber samples from the suspected virus-infected cucumber samples by the reverse transcriptase polymerase chain reaction method. The results showed that the cucumber samples with disease in the field were caused by a complex infection of multiple viruses including watermelon silver mottle virus. This is the first time that watermelon silver mottle virus was found to infect cucumbers under natural conditions in Hebei Province. Further studies have found that the virus sequences in cucumber plants infected with watermelon silver mottle virus in the field have certain complexity and diversity, and exchange and recombination may occur between different virus isolates. In the process of RNA virus replication, RNA as genetic material is prone to gene replacement, deletion and insertion. The genome sequence of the watermelon silver mottle virus introduced into Shijiazhuang, Hebei Province has changed significantly.

[0005] Based on the above findings, the present invention constructs an infectious clone, which has the same infectivity as the wild-type plant virus and exhibits the same infection symptoms after inoculation. The present invention uses primers to amplify the full-length sequence of the watermelon silver mottle virus genomic RNA prevalent in Shijiazhuang area in three segments, and constructs cDNA infectious clones corresponding to LRNA, M RNA and S RNA, which are applied to the inoculation system of watermelon silver mottle virus, and are used to screen resistant germplasm resources against watermelon silver mottle virus according to the virus expression in cucumber combined with the phenotype. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, there is provided a cucumber infectious nucleic acid derived from watermelon silver mottle virus, which is constructed using a primer set with the watermelon silver mottle virus genome as a template;

[0007] Wherein, the primer set comprises:

[0008] (1) Primers for cloning the full genome L RNA of watermelon silver mottle virus, the sequences of which are shown in SEQ ID NO.1-6;

[0009] (2) Primers used to clone the full genome M RNA of watermelon silver mottle virus, whose sequences are shown in SEQ ID NOs. 7-10;

[0010] (3) Primers used to clone the full genome S RNA of watermelon silver mottle virus, whose sequences are shown in SEQ ID NOs.11-14.

[0011] In certain embodiments, according to the cucumber infectious nucleic acid of the present invention, the nucleic acid comprises single-stranded L RNA, single-stranded M RNA and single-stranded S RNA from the whole genome of watermelon silver mottle virus, or comprises cDNA corresponding to these RNAs.

[0012] In certain embodiments, according to the cucumber infectious nucleic acid described in the present invention, the infectious cDNA corresponding to the L RNA has the nucleotide sequence shown in SEQ ID NO.15, the infectious cDNA corresponding to the M RNA has the nucleotide sequence shown in SEQ ID NO.16, and the infectious cDNA corresponding to the S RNA has the nucleotide sequence shown in SEQ ID NO.17.

[0013] The second aspect of the present invention provides a method for constructing an infectious nucleic acid, which comprises the following steps: using a pCB301-CH vector, integrating the complete genome of watermelon silver mottle virus to generate a recombinant expression vector, constructing a recombinant virus, and cloning it using Escherichia coli competent cells.

[0014] The third aspect of the present invention provides a biological material containing the infectious nucleic acid, wherein the biological material further includes an expression cassette, an expression vector, a cloning vector, a host cell and / or an engineered bacterium.

[0015] The fourth aspect of the present invention provides the use of the infectious nucleic acid in cucumber cultivation.

[0016] In certain embodiments, the use according to the present invention includes use in plant breeding and / or resistant germplasm screening.

[0017] The fifth aspect of the present invention provides an inoculation system for watermelon silver mottle virus, which comprises the infectious nucleic acid.

[0018] The sixth aspect of the present invention provides a method for screening plants resistant to watermelon silver mottle virus, which comprises the following steps: using the infectious nucleic acid of the present invention as a virus source, transforming it into Agrobacterium, infecting plant seedlings, and screening plants resistant to watermelon silver mottle virus based on the virus proliferation in the plant and the disease occurrence of the plant.

[0019] In certain embodiments, according to the method for screening plants resistant to watermelon silver mottle virus of the present invention, the plant seedlings are cucumber seedlings.

[0020] In the present invention, the primer set sequence used for segmental cloning of the watermelon silver mottle virus genome is as follows:

[0021] L RNA-F1: 5'-AGAGCAATCGTGCAACAAATAAGAA-3', SEQ ID NO.1;

[0022] L RNA-R1: 5'-AATGTAGATCCTGTGATAGGGCA-3', SEQ ID NO.2;

[0023] L RNA-F2: 5'-ATTCTAACTTGCGCTAACACAAGC-3', SEQ ID NO.3;

[0024] L RNA-R2: 5'-TTCCCTTGAAGCCAGTTCATGG-3', SEQ ID NO.4;

[0025] L RNA-F3: 5'-ACTTCCTCTGACAGAGACTTCTATC-3', SEQ ID NO.5;

[0026] L RNA-R3: 5'-AGAGCAATCGAGCAACAAAATAAAAGT-3', SEQ IDNO.6;

[0027] M RNA-F1: 5'-AGAGCAATCGGTGCGCCAAT-3', SEQ ID NO.7;

[0028] M RNA-R1: 5'-CAGAAAATTCTCACAGGGAACAT-3', SEQ ID NO.8;

[0029] M RNA-F2: 5'-ATGTTCCTGTGAGAATTTTCTG-3', SEQ ID NO.9;

[0030] M RNA-R2: 5'-CGACTCCTATAGGGCGATTG-3', SEQ ID NO.10;

[0031] S RNA-F1: 5'-AGAGCAATCGAGGCAAATAATAATTCT-3', SEQ ID NO.11;

[0032] S RNA-R1: 5'-ACAATAGGGCTCTTCAATTGTGATT-3', SEQ ID NO.12;

[0033] S RNA-F2: 5'-AATCACAATTGAAGAGCCCTATT-3', SEQ ID NO. 13;

[0034] S RNA-R2: 5'-AGAGCAATCGAGGCGCTAATAAAA-3', SEQ ID NO. 14.

[0035] The present invention also provides a complete genome of watermelon silver mottle virus, which includes three single-stranded RNAs (L, M and S RNA), wherein the L RNA is cloned using the primers of the above-mentioned L RNA, and the nucleotide sequence is shown in SEQ ID NO.15; the M RNA is cloned using the primers of the above-mentioned M RNA, and the nucleotide sequence is shown in SEQ ID NO.16; the S RNA is cloned using the primers of the above-mentioned S RNA, and the nucleotide sequence is shown in SEQ ID NO.17.

[0036] The present invention further provides application of the whole genome of watermelon silver mottle virus in watermelon silver mottle virus detection.

[0037] The present invention further provides a qPCR primer for detecting watermelon silver mottle virus, and the primer sequence is as follows:

[0038] YN-F: 5'-TGACGTTGGACCTGAAGACT-3', the sequence of which is as SEQ No. 18;

[0039] YN-R: 5'-TGCTAGTCCCCTTATGCTGG-3', its sequence is as SEQ No.19.

[0040] The present invention also provides watermelon silver mottle virus genome L RNA, M RNA and S RNA infectious cDNAs, wherein the infectious cDNA corresponding to the LRNA is pCB301-CH-WSMoV-L, and the nucleotide sequence is shown in SEQ ID NO.15; the infectious cDNA corresponding to the M RNA is pCB301-CH-WSMoV-M, and the nucleotide sequence is shown in SEQ ID NO.16; the infectious cDNA corresponding to the S RNA is pCB301-CH-WSMoV-S, and the nucleotide sequence is shown in SEQ ID NO.17.

[0041] The invention also provides a method for constructing infectious cDNA of L RNA, M RNA and S RNA of the watermelon silver mottle virus genome, comprising the following steps: using a low-copy high-stability pCB301-CH vector, integrating the complete genome of the watermelon silver mottle virus to generate pCB301-CH-WSMoV-L, pCB301-CH-WSMoV-M and pCB301-CH-WSMoV-S vectors, constructing recombinant viruses, and using Stbl3 Escherichia coli competent cells to reduce the probability of erroneous recombination.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention targets a specific watermelon silver mottle virus found in Shijiazhuang area, uses segmented primers to respectively amplify and integrate L RNA, M RNA and S RNA of the watermelon silver mottle virus genome, constructs pCB301-CH-WSMoV-L, pCB301-CH-WSMoV-M and pCB301-CH-WSMoV-S infectious full-length cDNA clones, and applies them to the screening of cucumber resistant germplasm resources. Compared with the prior art, the invention has simple operation, high inoculation incidence rate and high identification accuracy, and can quickly and accurately identify the disease resistance of cucumber germplasm resources at the seedling stage, thereby improving breeding efficiency, saving breeding costs and accelerating the breeding process. The identification method can be applied to the disease resistance identification of cucumber germplasm resources, various parents, hybrids and other materials.

[0044] In addition, the infectious cDNA clone prepared by the present invention has low cost, simple operation and convenient and stable storage, avoiding the degradation of RNase during in vitro transcription. Currently, there is a lack of available infectious clones of watermelon silver mottle virus in research and production. The present invention effectively solves the technical problem of being unable to screen cucumber germplasm resources that are resistant to watermelon silver mottle virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Cucumber samples infected with watermelon silver mottle virus in the field at Dahe Experimental Base, Shijiazhuang City, Hebei Province (longitude 114.39°, latitude 38.13°).

[0046] Figure 2 Gel image of segmented amplification of the full-length sequence of watermelon silver mottle virus in Shijiazhuang City, Hebei Province; Marker: DNA Marker; L-1: amplification of the first segment of the L RNA chain; L-2: amplification of the second segment of the L RNA chain; L-3: amplification of the third segment of the L RNA chain; M-1: amplification of the first segment of the M RNA chain; M-2: amplification of the second segment of the M RNA chain; S-1: amplification of the first segment of the S RNA chain; S-2: amplification of the second segment of the S RNA chain.

[0047] Figure 3 Infectious clonal inoculation method, AB: seed germination; C: explant treatment; D: co-cultivation; E: transplanting; F: infection with watermelon silver mottle virus.

[0048] Figure 4 Establishment of the identification technology system for Agrobacterium tumefaciens inoculation of watermelon silver mottle virus infective clones; C and D are sensitivity comparisons (M: DNA Marker; 1-10: copy number 3.77×10 10 -3.77×10 1 copies·μL -1 WSMoV-CP-247 plasmid samples, N: negative control). A and C: qPCR detection; B: standard curve; D: conventional PCR detection.

[0049] Figure 5 Symptoms of cucumber infected with watermelon silver mottle virus; Note: CK refers to leaves of cucumber germplasm that are not infected with watermelon silver mottle virus, WSMoV-15dpi and WSMoV-30dpi refer to leaves infected with watermelon silver mottle virus 15 days and 30 days later. DETAILED DESCRIPTION

[0050] 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.

[0051] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0052] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. 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 related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0053] Materials used in the following examples: Agrobacterium-infectious clones of watermelon silver mottle virus and related cucumber seed resources are available to the public from the Biotechnology Laboratory of the Institute of Economic Crops, Hebei Academy of Agricultural and Forestry Sciences to repeat this experiment. Unless otherwise specified, the watermelon silver mottle virus mentioned herein refers to the Hebei strain of watermelon silver mottle virus.

[0054] As used herein, the term "infectious clone" refers to an infectious cDNA of an RNA virus or a nucleic acid that produces infectious RNA by in vitro transcription.

[0055] Example 1

[0056] This example shows the cloning of the full-length sequence of watermelon silver mottle virus in Shijiazhuang City, Hebei Province.

[0057] Extract plant virus samples collected in Shijiazhuang City, Hebei Province ( Figure 1 ) RNA was reverse transcribed to synthesize cDNA. Based on the non-cucumber-infectious watermelon silver mottle virus isolate viruses included in GeneBank (NC_003832.1 / NC_003841.1 / NC_003843.1), segmented primers were designed to amplify the sequence of the watermelon silver mottle virus Shijiazhuang isolate ( Figure 2 ), and the PCR primer sequences are shown in SEQ ID NO. 1 to SEQ ID NO. 14. The amplified sequences were sequenced and analyzed to be different from the known sequences.

[0058] After PCR amplification, the target fragment was recovered, connected to the sequencing vector and transformed into E. coli, and a single colony was picked for PCR detection. A single colony with the correct band size was picked and sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequence was then spliced, where LRNA was 8915 bp long, the sequence was shown in SEQ ID NO.15, MRNA was 4927 bp long, the sequence was shown in SEQ ID NO.16, and S RNA was 3602 bp long, the sequence was shown in SEQ ID NO.17.

[0059] Example 2

[0060] This example shows the construction of recombinant pCB301-CH-WSMoV-L, M and S infectious cloning vectors.

[0061] Based on the low-copy, high-stability pCB301-CH vector, the complete genome of watermelon silver mottle virus was integrated to generate pCB301-CH-WSMoV-L, pCB301-CH-WSMoV-M and pCB301-CH-WSMoV-S vectors, and recombinant viral vectors were constructed. Stbl3 Escherichia coli competent cells were used to reduce the probability of erroneous recombination, so that the full-length cDNA clone of watermelon silver mottle virus could be stably replicated in Escherichia coli and form Agrobacterium-mediated infectious clones. The specific process is as follows:

[0062] According to the full-length sequence of the plant virus samples collected in Shijiazhuang City, Hebei Province, and the homology arm sequence of the linearized vector, the segmented homologous recombination primers were designed, and the high-fidelity enzyme of Quanshijin Company was used. FastPfu Fly DNA Polymerase was used to perform PCR amplification using cDNA reverse transcribed from WSMoV-L RNA, WSMoV-M RNA, and WSMoV-S RNA as templates. The PCR products were subjected to 1% agarose gel electrophoresis and recovered by gel excision. The pCB301-CH vector was double-digested with StuⅠ and SmaⅠ, and the linearized vector fragment was excised and recovered according to -Basic Seamless Cloning and Assembly Kit was used to establish a homologous recombination reaction system. The recombinant product was transformed into Stbl3 Escherichia coli competent cells, and a single colony was picked for PCR detection. A single colony of the correct size was picked and sent to Sangon Biotech Co., Ltd. for sequencing. After sequencing verification, the plasmid was extracted and transformed into Agrobacterium EHA105, and the strain was stored at -80℃.

[0063] Example 3

[0064] This example shows the establishment of a cucumber inoculation system of the Hebei strain of watermelon silver mottle virus.

[0065] The constructed Agrobacterium watermelon silver mottle virus infectious cDNA clone was used as the virus source to infect cucumber varieties 9930, T8163, T8164 and T8165, observe the symptoms of the plants, and establish a stable watermelon silver mottle virus inoculation system. The YN-F / R primers for the nucleocapsid protein (N) gene of the watermelon silver mottle virus provided by the present invention were used to measure the virus copy number in the plant after inoculation, and the disease resistance of the variety was determined according to the virus content in the plant and the disease condition of the plant. The specific process is as follows:

[0066] (1) Seed sterilization: Select complete and full cucumber seeds and place them in a sterilized triangular flask. Sterilize with 75% ethanol for about 1 minute, gently shake them to make them fully contact, and then rinse them three times with sterile water. Rinse with 2-3% NaClO for 10 minutes, gently shake them to make them fully contact, and then rinse them 3-5 times with sterile water. Transfer the seeds to a sterilized culture dish, absorb the surface moisture with filter paper, and use tweezers to evenly spread the seeds on the MS culture medium. Pre-culture at 25-28℃ in the dark for about 3 days (the germination speed of seeds of different cucumber varieties is inconsistent, and the next step can be carried out when 1 / 2-1 / 3 of the cotyledons are exposed from the seed coat).

[0067] (2) Culture the Agrobacterium tumefaciens that is infectious to watermelon silver mottle virus in liquid LB medium overnight, centrifuge and discard the supernatant to obtain the bacteria, and resuspend them in infection buffer (10mM 3-(N-morpholino)ethane sulfonic acid (MES), 10mMMgCl2, 200μM acetosyringone (AS) and 5% (w / v) sucrose) for later use. Use the heated tweezers to take the germinated seeds and place them in a culture dish. Cut off the 1 / 3 cotyledonary node away from the hypocotyl, and the remaining part is used as the explant to be transformed. Use a blade to make several wounds on the explant to facilitate infection. Transfer the explants to the prepared infection buffer and soak them for 15 minutes. During this period, gently shake the explants to ensure full contact with the bacterial solution, and then use filter paper to absorb the surface moisture. Evenly place the explants on MS (200μM AS) co-culture medium and culture them in the dark. After 2 days, plant them in sterile nutrient soil, cover them to keep them moist until they survive, and culture them in an incubator away from insects to continuously observe the symptoms ( Figure 3 ).

[0068] Example 4

[0069] Premier 5.0 software was used to perform real-time fluorescence quantitative PCR using primers YN-F / R. The cDNA of the cucumber sample positive for watermelon silver mottle virus was used as a template for PCR amplification using YN-F / R, and the correct band was cut and recovered. The recovered PCR product was ligated to -Blunt Simple Cloning Vector. The clones with correct sequencing were selected for bacterial culture and plasmid extraction. The concentration of the extracted plasmid was 168.4 ng / μL. The copy number of the recombinant plasmid was calculated to be 3.77×10 10 copies / μL. Select a dilution gradient of 10 -3 -10 -7 The plasmid standard was used to prepare the standard curve. The corresponding dilution gradient was 10 -3 -10 -7 The copy number of the plasmid standard is 3.77×10 7 -3.77×10 3 copies / μL( Figure 4 A).

[0070] According to the obtained amplification curve, the concentration of the template used is proportional to the number of cycles that can detect the fluorescence signal within a certain range. When the concentration of the template exceeds a certain value, the Ct value is too small, causing the fluorescence intensity to enter the plateau phase prematurely, which will make the detection value not conform to the linear relationship; on the contrary, when the Ct value exceeds 35, it does not conform to the linear relationship. Therefore, in the experiment, the Ct values ​​of the plasmid standard samples with 5 concentration gradients that are all within the measurable effective range are selected to prepare the standard curve. Figure 4 B) The slope of the standard curve was -3.7878, and the relative R was 0.9994. 2 This result shows that the prepared standard curve has a good linear relationship, and the regression equation is y=-4.093x+40.614 (y represents the Ct value, x represents the logarithmic value of the plasmid copy number). This regression equation can be used to calculate the starting copy number of the unknown sample to be tested.

[0071] The YN-F / R primer sequences are as follows:

[0072] YN-F: 5'-TGACGTTGGACCTGAAGACT-3', the sequence of which is as SEQ No. 18;

[0073] YN-R: 5'-TGCTAGTCCCCTTATGCTGG-3', its sequence is as SEQ No.19.

[0074] The selected copy number is 3.77×10 1 -3.77×10 10 The YN plasmid sample with 10 copies / μL was used as a template, and YN-F / R primers were used for RT-qPCR and conventional RT-PCR reactions. The results showed that the RT-qPCR detection method could detect a minimum of 3.77×10 2copies / μL of plasmid sample ( Figure 4 C), conventional RT-PCR detection method can only detect 3.68×104 copies / μL of plasmid samples ( Figure 4 D) shows that the sensitivity of RT-qPCR detection using YN-F / R primers is about 100 times higher than that of conventional RT-PCR detection method.

[0075] Example 5

[0076] This example shows the symptoms of cucumber infected with watermelon silver mottle virus and the detection of watermelon silver mottle virus content.

[0077] The 9930, T8163, T8164 and T8165 used in this example are different codes for cucumber varieties; the 9930, T8163, T8164 and T8165 cucumber varieties used are all derived from germplasm resources preserved in this laboratory.

[0078] The inoculation system of Example 3 was used to infect cucumber varieties 9930, T8163, T8164 and T8165, and the YN-F / R primers were used to detect the virus content in the inoculated plants. The experimental results are shown in Tables 1 and Figure 5 As shown in the figure, 9930, T8163, T8164 and T8165 can all be infected with the watermelon silver mottle virus 15 days after infection, and the disease is more serious after 30 days. The symptoms of leaf disease are basically the same: leaves are wrinkled, deformed, yellowed, and faded.

[0079] Table 1 Virus copy numbers of different cucumber germplasms after infection with watermelon silver mottle virus

[0080]

[0081] The method of the present invention can be used to screen the disease resistance of cucumbers in the cucumber seedling stage, and the cucumbers are infected quickly with high efficiency and obvious symptoms, which is helpful for efficiently screening cucumber varieties resistant to watermelon silver mottle virus disease. The method is simple to operate, has high inoculation efficiency, and has stable symptoms, and can be used for large-scale inoculation.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cucumber infectious nucleic acid derived from watermelon silver mottle virus, characterized in that: It was constructed using a primer set with the watermelon silver mottle virus genome as a template; Wherein, the primer set comprises: (1) Primers for cloning the full genome L RNA of watermelon silver mottle virus, the sequences of which are shown in SEQ ID NO.1-6; (2) Primers used to clone the full genome M RNA of watermelon silver mottle virus, whose sequences are shown in SEQ ID NOs. 7-10; (3) Primers used to clone the full genome S RNA of watermelon silver mottle virus, whose sequences are shown in SEQ ID NOs.11-14.

2. The cucumber infectious nucleic acid according to claim 1, characterized in that The nucleic acid comprises single-stranded L RNA, single-stranded M RNA and single-stranded S RNA from the whole genome of watermelon silver mottle virus, or comprises cDNA corresponding to these RNAs.

3. The cucumber infectious nucleic acid according to claim 1, characterized in that The infectious cDNA corresponding to the L RNA has the nucleotide sequence shown in SEQ ID NO.15, the infectious cDNA corresponding to the M RNA has the nucleotide sequence shown in SEQ ID NO.16, and the infectious cDNA corresponding to the S RNA has the nucleotide sequence shown in SEQ ID NO.

17.

4. The method for constructing an infectious nucleic acid according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: using pCB301-CH vector, integrating the complete genome of watermelon silver mottle virus to generate a recombinant expression vector, constructing a recombinant virus, and cloning it with competent Escherichia coli cells.

5. A biological material containing the infectious nucleic acid according to any one of claims 1 to 3, characterized in that: The biological material further includes an expression cassette, an expression vector, a cloning vector, a host cell and / or an engineered bacterium.

6. Use of the infectious nucleic acid according to any one of claims 1 to 3 in cucumber cultivation.

7. The use according to claim 6, characterized in that: The application includes application in plant breeding and / or resistant germplasm resource screening.

8. A watermelon silver mottle virus inoculation system, characterized in that: Comprising the infectious nucleic acid according to any one of claims 1-3.

9. A method for screening plants resistant to watermelon silver mottle virus, characterized in that: The method comprises the following steps: using the infectious nucleic acid described in any one of claims 1 to 3 as a virus source, transforming it into Agrobacterium, infecting plant seedlings, and screening plants resistant to watermelon silver mottle virus according to the virus proliferation in the plants and the disease conditions of the plants.

10. The method for screening plants resistant to watermelon silver mottle virus according to claim 9, characterized in that: The plant seedlings are cucumber seedlings.

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