Construction method and application of tea tree reticulate spot virus infectious clone and VIGS vector

Abstract

The invention relates to an infectious clone of tea tree reticulate spot virus and a method for constructing a VIGS vector thereof, and its application, which belong to the field of genetic engineering technology. The infectious clone comprises three full-length cDNA recombinant plasmids of tea tree reticulate spot virus (TPLPV), namely pTPLPV1, pTPLPV1 and pTPLPV3. pTPLPV3 can be used to insert exogenous gene fragments to form a VIGS vector of TPLPV. By Agrobacterium-mediated method, pTPLPV3-VIGS is inoculated alone or mixed with the first two chains for infection. The NbPDS gene of the model plant tobacco can be silenced and an albino silent phenotype can be produced. Infection alone can silence the CsPDS gene of the tea tree and produce an albino silent phenotype. The present invention transforms the tea tree virus TPLPV into an infectious clone and a VIGS vector for the first time, providing a tool carrier and technical support for studying the function of the viral protein, the pathogenic mechanism and the construction and transformation of the silencing vector.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a tea reticular spot virus infectious clone and a VIGS vector construction method and application thereof. Background Art

[0002] Tea (Camellia sinensis) is an important economic crop in my country. With the publication of its genome sequence, tea genetic research has entered the era of functional genomics. However, due to the abundance of secondary metabolites such as polyphenols in tea plants and the difficulty of in vitro regeneration, research on tea genetic transformation has been slow. While there have been reports of successful tea transgenics, a stable tea genetic transformation system has yet to be established, severely hindering the identification of homologous gene functions in tea plants.

[0003] Developed based on the antiviral mechanisms of plants, virus-induced genesilencing (VIGS) is a post-transcriptional gene silencing technology that does not rely on plant genetic transformation systems. A viral silencing vector carrying a target gene fragment replicates within the host after invading the host, producing dsRNA, which is then cleaved into 21-24 nt siRNA and degraded into single-stranded RNA, silencing the target gene under the mediation of single-stranded RNA. Since its introduction in 1995, this technology has been widely used. Currently, the viruses that have been transformed into VIGS vectors mainly include RNA viruses, DNA viruses, and satellite viruses. It is widely used in research on the functional identification of genes related to growth and development, signal transduction, metabolic pathways, and stress resistance in herbaceous and woody plants. However, there are currently no reports of the successful application of VIGS technology in tea plants, nor is there a precedent for using tea plant viruses to transform into VIGS vectors.

[0004] In 2018, Hao et al. from the inventor's research group first identified viruses in tea plants. Among them, Tea plant line pattern virus (TPLPV) belongs to the Bromoviridae family and the Ilarvirus genus. Its genome size is approximately 7.7kb, containing three positive-sense single-stranded RNAs and a total of four ORFs. RNA1 encodes a methyltransferase-helicase with a molecular weight of 117kDa. RNA2 encodes an RNA polymerase with a molecular weight of 80kDa. RNA3 encodes a movement protein (MP) and a coat protein (CP), with molecular weights of 32kDa and 23kDa, respectively. Viruses of the same family as this virus, such as Brome mosaic virus (BMV) and Cucumber mosaic virus (CMV), have been successfully transformed into VIGS vectors. Therefore, in order to transform tea tree virus into VIGS vector and provide tool vectors and technical support for the establishment of tea tree VIGS system, it is urgently necessary to use tea tree virus for the construction and application of infectious cloning vectors and VIGS vectors. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for the construction method and application of the infectious clone of tea tree reticular spot virus and its VIGS vector.

[0006] The present invention is specifically implemented through the following technical solutions:

[0007] The first aspect of the present invention provides a tea tree web spot virus infectious cloning vector, which is constructed from the cDNA sequence of tea tree web spot virus TPLPV and the modified pCAMBIA1300 plasmid;

[0008] The cDNA sequence of the tea tree web spot virus TPLPV includes TPLPV1, TPLPV2 and TPLPV3, and the sequences of TPLPV1, TPLPV2 and TPLPV3 are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively;

[0009] The modified pCAMBIA1300 plasmid contains a 35S promoter and a NOS terminator. The sequences of the 35S promoter and the NOS terminator are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

[0010] A second aspect of the present invention provides a method for preparing a tea tree web spot virus infectious cloning vector, comprising the following steps:

[0011] (1) Based on the TPLPV genome information, cDNA sequences corresponding to the three components of TPLPV were artificially synthesized, namely TPLPV1, TPLPV2, and TPLPV3;

[0012] (2) TPLPV1, TPLPV2, and TPLPV3 were constructed into the modified pCAMBIA1300 by homologous recombination to obtain pTPLPV1, pTPLPV2, and pTPLPV3, respectively;

[0013] (3) GFP sequences were incorporated into the 3′ end of the MP and CP of pTPLPV3 to further evaluate the expression efficiency of the vectors used for TPLPV;

[0014] (4) Using the Agrobacterium-mediated injection inoculation method, pTPLPV1, pTPLPV2, and pTPLPV3 recombinant viral plasmids were mixed in equal proportions, or pTPLPV3 viral recombinant plasmid was used alone to infect tobacco plants at the 3-5 leaf stage.

[0015] Furthermore, when synthesizing TPLPV3 in step (1), SacI and BamHI restriction sites are added before the stop codon of the first ORF, i.e., the mobile protein, and SpeI and NcoI restriction sites are added before the stop codon of the second ORF, i.e., the coat protein.

[0016] The third aspect of the present invention provides the use of the above-mentioned tea reticulate spot virus infectious cloning vector in the study of tea reticulate spot virus protein function, analysis of viral gene pathogenicity mechanism, and construction and transformation of silencing vectors.

[0017] A fourth aspect of the present invention provides an Agrobacterium strain carrying a tea web spot virus infectious clone, which is obtained by introducing the tea web spot virus infectious clone vector according to claim 1 into the Agrobacterium strain GV3101 through the freeze-thaw method.

[0018] The fifth aspect of the present invention provides the use of the above-mentioned Agrobacterium strain in the study of the function of tea leaf spot virus protein, the analysis of the pathogenic mechanism of viral genes, and the construction and transformation of silencing vectors.

[0019] The sixth aspect of the present invention provides a tea tree reticular spot virus gene silencing vector, which includes pTPLPV1, pTPLPV2 and pTPLPV3-VIGS;

[0020] The pTPLPV3-VIGS is obtained by constructing the target gene fragment to be silenced onto the pTPLPV3 linearized vector after NcoI digestion through homologous recombination method;

[0021] The pTPLPV1, pTPLPV2 and pTPLPV3 are prepared by constructing the cDNA sequence of the tea tree web spot virus TPLPV into the modified pCAMBIA1300 plasmid;

[0022] The cDNA sequence of the tea tree web spot virus TPLPV includes TPLPV1, TPLPV2 and TPLPV3, and the sequences of TPLPV1, TPLPV2 and TPLPV3 are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively;

[0023] The modified pCAMBIA1300 plasmid contains a 35S promoter and a NOS terminator. The sequences of the 35S promoter and the NOS terminator are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

[0024] Furthermore, SpeI and NcoI restriction sites were added before the termination codon of the second ORF of the pTPLPV3, namely the coat protein.

[0025] The seventh aspect of the present invention provides the use of the above-mentioned tea web spot virus gene silencing vector in gene silencing in tobacco and tea plants.

[0026] Furthermore, through the Agrobacterium-mediated method, pTPLPV3-VIGS was inoculated alone or mixed with pTPLPV1 and pTPLPV2 chains, which could silence the NbPDS gene of the model plant tobacco and produce an albino silent phenotype. pTPLPV3-VIGS alone could silence the CsPDS gene of tea plant and produce an albino silent phenotype.

[0027] Furthermore, the exogenous gene fragments inserted in the present invention are the Nicotiana benthamiana phytoene dehydrogenase (Phytoenedesaturase, PDS), Mg-chelatase I subunit (Mg-chelatase I subunit, ChlH) and Camellia sinensis phytoene desaturase (Phytoene desaturase, PDS) genes. After the above genes are silenced, the plants will show an albinism or chlorosis phenotype.

[0028] The present invention provides for the first time the infectious cloning vector, VIGS vector, construction method and application of tea tree virus TPLPV, providing tool vectors and technical support for studying the protein function, pathogenic mechanism and construction and modification of silencing vectors of tea tree virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1: Fluorescence observation results 2 days after inoculation of tobacco with pTPLPV-GFP. (a) p1300-GFP; (b) pTPLPV3-MPGFP; (c) pTPLPV3-CPGFP.

[0030] Figure 2 : Phenotypic observations of pTPLPV infectious clones 15 days after inoculation into tobacco. (a) Control; (b) pTPLPV3; (c) pTPLPV1+2+3.

[0031] Figure 3 Phenotypic observations 46 days after inoculation of tobacco with pTPLPV-VIGS. (a) Control; (b) pTPLPV1+2+3; (c) pTPLPV1+2+3-CPNbPDS300F; (d) pTPLPV3-CPNbPDS300F.

[0032] Figure 4 : NbPDS expression analysis results 46 days after pTPLPV-VIGS inoculation in tobacco. Different lowercase letters represent significant differences among different treatments (P<0.05).

[0033] Figure 5 Phenotypic observations of tea plant cuttings 285 days after inoculation with pTPLPV-VIGS. (a) Control; (b) pTPLPV3; (c) pTPLPV3-CPNbPDS300F.

[0034] Figure 6 : Analysis of CsPDS expression in leaves of tea cuttings 285 days after inoculation with pTPLPV-VIGS. DETAILED DESCRIPTION

[0035] In the following examples, unless otherwise specified, the experimental methods used are conventional methods.

[0036] In the following examples, unless otherwise specified, the materials and reagents used can be obtained through commercial channels.

[0037] Example 1: Construction of TPLPV infectious cloning vector

[0038] Based on the published TPLPV genome information, which indicates that the tea tree web spot virus contains three positive-sense single-stranded RNA strands, the corresponding cDNA sequences for TPLPV were synthesized by Shanghai Huajin Biotechnology Co., Ltd. When synthesizing the third strand, SacI and BamHI restriction sites were added before the stop codon of the first ORF, the movement protein (MP), and SpeI and NcoI restriction sites were added before the stop codon of the second ORF, the coat protein (CP), to facilitate the insertion of exogenous gene fragments. The sequences of TPLPV1, TPLPV2, and TPLPV3 are shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively.

[0039] After synthesizing the cDNAs for each TPLPV component, they were constructed and incorporated into the modified pCAMBIA1300 vector via homologous recombination to generate pTPLPV1, pTPLPV2, and pTPLPV3, respectively. The modified pCAMBIA1300 vector contains the 35S promoter and NOS terminator. The cDNAs corresponding to each TPLPV component were placed 3' to the 35S promoter, driving expression of the respective components. The sequences of the 35S promoter and NOS terminator are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

[0040] Example 2: Construction of promoter-linked GFP recombinant vector

[0041] In order to further evaluate the expression efficiency of the vector for the target gene fragment, the green fluorescent protein (GFP) gene sequence was fused to the 3' end of the promoter. The GFP gene sequence is shown in SEQ ID NO.6.

[0042] Using the pCAMBIA1301 plasmid as a template, the primer pair 35S-SC-F1 / 35S-SOE-R1 was used for cloning and gel extraction to obtain the 35S promoter (346 bp). The target gene fragment was amplified using KOD-Plus-Neo high-fidelity polymerase (TOYOBO, Japan). The reaction system consisted of 10× KOD buffer (5 μL), dNTPs (5 μL), MgSO₄ (5 μL), forward primer (1 μL), reverse primer (1 μL), template (1 μL), KOD-Plus-Neo (1 μL), and ddH₂O (33 μL). The reaction procedure was as follows: 94°C for 3 min; 98°C for 10 s, annealing temperature Tm / 30 s, 68°C / (1 kb / 30 s), 35 cycles; 68°C for 7 min; and 4°C / ∞. The target gene fragment was recovered from the gel according to the instructions of AxyPrepTM DNA Gel Extraction Kit (Axygen, USA).

[0043] Using the p35SGFP vector as a template, the primer pair EGFP-SOE-F2 / EGFP-SC-R2 was used for cloning, and the GFP gene sequence (720 bp) was recovered by gel extraction. 35S was fused to GFP by SOE PCR to generate 35SGFP. Using the correctly sequenced 35SGFP plasmid as a template, the primer pair 35S-SC-F1 / EGFP-SC-R2 was used for cloning, and the 35SGFP gene (1066 bp) was recovered by gel extraction.

[0044] p1300NOS was double-digested with EcoRI and KpnI restriction endonucleases (Thermo Scientific, USA) using the following digestion system: plasmid (2-5 ng), Enzyme 1 (1 μL), Enzyme 2 (1 μL), 10× FD buffer (1 μL), and ddH2O (28 μL). Mix well and incubate at 37°C for 2-3 hours. Follow with gel electrophoresis, and recover the digested fragment.

[0045] Following the instructions for the seamless cloning kit (GBclonart, Suzhou Gene Co., Ltd., China), the target gene fragment was constructed into the linearized vector by homologous recombination. The vector dosage was 50-100 ng, with a target fragment to vector molar ratio of 2:1-3:1. The reaction system was as follows: GBclonart recombinant buffer (15 μL), linearized vector + PCR fragment + ddH2O (5 μL total). Mix thoroughly and place in a 45°C metal bath or PCR instrument for 30 minutes to obtain the recombinant plasmid. Transfer to ice and set aside. 35SGFP was constructed into pCAMBIA1300 by homologous recombination to generate p1300-GFP.

[0046] Example 3: Construction of TPLPV-linked GFP recombinant vector

[0047] In order to further evaluate the expression efficiency of the vector used for TPLPV, GFP gene sequences were incorporated into the 3' end of MP and CP of pTPLPV3 respectively.

[0048] pTPLPV3-MPGFP: Using the p35SGFP plasmid as a template, primers TPLPV3-MPGFP-F / TPLPV3-MPGFP-R were used for cloning, and GFP (720 bp) was recovered by gel extraction. pTPLPV3 was digested with SacI, the linearized vector was recovered by gel extraction, and GFP was constructed into pTPLPV3 by homologous recombination to obtain pTPLPV3-MPGFP.

[0049] pTPLPV3-CPGFP: Using the p35SGFP plasmid as a template, primers TPLPV3-CPGFP-F / TPLPV3-CPGFP-R were used for cloning, and GFP (720 bp) was recovered by gel extraction. pTPLPV3 was digested with NcoI, the linearized vector was recovered by gel extraction, and GFP was constructed into pTPLPV3 by homologous recombination to obtain pTPLPV3-CPGFP.

[0050] Table 1 Primers for construction of p1300-GFP and TPLPV-GFP vectors

[0051]

[0052] Note: Double underlines indicate sequences homologous to the vector.

[0053] All TPLPV recombinant viral vectors involved in the present invention were sequenced and verified.

[0054] The obtained infectious clones and recombinant viral vectors with GFP sequence were introduced into Agrobacterium strain GV3101 by freeze-thaw method. The main steps are as follows: (1) Thaw Agrobacterium competent cells on ice. (2) Add 0.4-0.5μg plasmid to the freshly dissolved Agrobacterium competent cells (100μL) and place on ice for 30 minutes. (3) Quickly freeze in liquid nitrogen for 5 minutes, then transfer to a 37℃ metal bath for reaction for 5 minutes. (4) Add 500μL LB liquid culture medium without antibiotics to the clean bench and place in a shaker at 28℃, 200rpm, for 2-4 hours. (5) Plate: LB plates containing kanamycin (kan, 50μg / mL) and rifampicin antibiotics (rif, 50μg / mL) are used to plate. Take about 100μL of liquid and plate. (6) Pick a single clone for bacterial liquid PCR verification.

[0055] Example 4: Preparation and inoculation of Agrobacterium inoculum

[0056] The inoculum preparation method is as follows: (1) Take a 50 mL centrifuge tube, add LB liquid medium (containing 50 μg / mL kan and 50 μg / mL rif), and shake at 28°C and 200 rpm until the bacterial solution OD 600 = about 1.0. (2) Mix the bacterial solution and IM and shake the mixture: bacterial solution (1 mL), IM (25 mL, prepared as follows: distilled water 350 mL, 5-morpholineethanesulfonic acid 5.137 g, glucose 2.632 g, sodium dihydrogen phosphate 0.164, dilute to 500 mL, adjust pH to 5.6-5.7, and sterilize by autoclaving), AB salts (1.3158 mL, prepared as follows: NH4Cl 2 g, MgSO4·7H2O 0.6 g, KCl 0.3 g, CaCl2 0.02 g, FeSO4·7H2O 5 mg, dilute to 100 mL), 50 mg / mL kan (1 μL / 1 mL liquid), 200 mM acetosyringone (1 μL / 1 mL liquid). Shake at 28°C, 200 rpm until the bacterial solution OD reaches 0. 600 = about 1.0. (3) After centrifugation at 5000rpm for 10min, resuspend with the same volume of MES buffer (10mM MgCl2, 10mM MES pH5.7, 100μM acetosyringone) and repeat the centrifugation once. When only one component is used for inoculation, resuspend with the same volume of MES; for multiple components, such as three components, resuspend each component with 1 / 3 MES and then mix them equally. Add acetosyringone to make the final concentration of acetosyringone in the inoculum reach 200μM. (4) Place at 22-25℃ for 3-5h. Preparation is complete.

[0057] The injection inoculation method is as follows: (1) Use a 1 mL syringe to draw up the bacterial solution and use the needleless injection method to inoculate tobacco plants at the 3-5 leaf stage. The inoculated material is placed in the dark for 24 hours and then transferred to an artificial climate chamber for incubation (25°C, 16 hours light / 8 hours dark). (2) After inoculating tobacco and tomatoes with the infectious clone vector, take pictures regularly to observe the symptoms of the plants. 48 hours after inoculating tobacco with the recombinant viral vector fused with the GFP sequence, observe GFP fluorescence using a laser confocal microscope.

[0058] The vacuum injection method for inoculating tea seedlings is as follows: (1) Take a half-year-old tea cutting, wash the seedling with clean water, and wipe off the surface moisture. (2) Place the above-ground part of the seedling upside down in a beaker filled with inoculation solution. Place the beaker in a glass vacuum cylinder connected to a circulating water vacuum pump and inoculate at -0.08 MPa for 15 minutes. The inoculated material is placed in the dark for 48 hours and then transferred to an artificial climate chamber for incubation (25°C, 12 hours light / 12 hours dark).

[0059] p1300-GFP, pTPLPV3-MPGFP and pTPLPV3-CPGFP were inoculated into tobacco plants by Agrobacterium-mediated injection. After 2 days, green fluorescence ( Figure 1 ), indicating that pCAMBIA1300 can effectively express target genes including TPLPV.

[0060] By Agrobacterium-mediated injection inoculation, Nicotiana benthamiana was inoculated with pTPLPV3 (the third chain alone) and pTPLPV1+2+3 (three chains mixed in equal proportions). After 15 days, different degrees of chlorotic reticulate symptoms were observed on the veins and leaves of the inoculated area ( Figure 2 The results indicate that the infectious cloning vector currently constructed with pCAMBIA1300 as the vector backbone can produce disease symptoms in the area where Nicotiana benthamiana is inoculated, and that the third chain alone can also be effective.

[0061] Example 5: Tea tree reticularia virus VIGS vector and its construction method and application

[0062] RNA extraction from tobacco and tea plants

[0063] RNA was extracted from tobacco leaves using the EASY-DO Plant Total RNA Rapid Extraction Kit (EASY-DO, China), and from tea leaves using the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen, China). RNA concentration and purity were determined using a NanoDrop 2000 Micro-Nucleic Acid Quantifier (Thermo Fisher Scientific, USA), and RNA integrity was verified by 1% agarose gel electrophoresis.

[0064] Reverse transcription and cDNA synthesis

[0065] RNA was reverse transcribed into cDNA according to the PrimerScript RT Reagent Kit (Takara, Japan) instructions and diluted 10-fold for use as a template for gene cloning. The cDNA synthesis system was as follows: 5× gDNA Eraser Buffer (2 μL), gDNA Eraser (0.8 μL), RNA (1 μg), and ddH2O (made up to 10 μL with water). After incubation at 42°C for 2 minutes, 10 μL of the following reaction solution was added to each sample: 5× Primer Script Buffer 2 (4 μL), Primer Script RT Enzyme Mix 1 (1 μL), RT Primer Mix (1 μL), and ddH2O (4 μL). The solution was mixed and placed in a PCR instrument for incubation at 37°C for 15 minutes and then at 85°C for 5 seconds to generate cDNA, which was then placed on ice and used until ready for use.

[0066] Example 6: NbPDS, NbCH1H and CsPDS gene cloning

[0067] Gene cloning was performed using KOD-Plus-Neo high-fidelity polymerase (TOYOBO, Japan). Using tobacco cDNA as a template, the primer pair NbPDS-F / NbPDS-R cloned the NbPDS gene (1761 bp). The primer pair NbChlH-F / NbChlH-R cloned the NbChlH gene fragment (1037 bp). Using tea plant cDNA as a template, the primer pair CsPDS-F / CsPDS-R cloned the CsPDS gene (1749 bp).

[0068] Table 2 Primers for cloning NbPDS, NbChlH and CsPDS genes

[0069]

[0070] Example 7: TPLPV-VIGS vector construction

[0071] pTPLPV3-CPNbPDS300F: Using the NbPDS plasmid as a template, a 300-bp NbPDS fragment was cloned using the primer pair NbPDS300-F / NbPDS300-R and gel-recovered to obtain NbPDS300. pTPLPV3 was digested with NcoI, the linearized vector was recovered from the gel, and NbPDS300 was inserted into the pTPLPV3 CP before the stop codon by homologous recombination to generate pTPLPV3-CPNbPDS300F.

[0072] pTPLPV3-CPNbChlH327F: Using the NbChlH plasmid as a template, a 327 bp NbChlH fragment was cloned using the primer pair NbChlH327-F / NbChlH327-R. NbChlH327 was recovered from the gel. pTPLPV3 was digested with NcoI, the linearized vector was recovered from the gel, and NbChlH327 was inserted into the pTPLPV3 CP immediately before the stop codon by homologous recombination to generate pTPLPV3-CPNbChlH327F.

[0073] pTPLPV3-CPCsPDS300F: Using the CsPDS plasmid as a template, a 300 bp CsPDS fragment was cloned using the primer pair CsPDS300-F / CsPDS300-R and gel-recovered to obtain CsPDS300. pTPLPV3 was digested with NcoI, the linearized vector was recovered from the gel, and CsPDS300 was inserted into the CP of pTPLPV3 before the stop codon by homologous recombination to generate pTPLPV3-CPCsPDS300F.

[0074] Table 3 Primers for construction of pTPLPV-VIGS vector

[0075]

[0076] Note: Double underlines indicate sequences homologous to the vector.

[0077] After 46 days of injection of tobacco plants with pTPLPV3-CPCsPDS300F mixed with pTPLPV1 and pTPLPV2 in equal proportions, a certain degree of albinism phenotype could be observed in the middle of the plants. Compared with the control, the expression level of NbPDS decreased by 40% ( Figure 3 , Figure 4 When pTPLPV3-CPNbPDS300F was injected into tobacco alone, a more obvious albinism phenotype was produced. Compared with the control, the expression of NbPDS decreased by about 40%. Figure 3 , Figure 4 ).

[0078] pTPLPV-VIGS vacuum infiltration method inoculation of tea plants: pTPLPV3-CPCsPDS300F was inoculated alone into 'Zhongming No. 7' cuttings by vacuum infiltration injection. After 285 days, albino silencing phenotype could be observed near the veins of tea leaves. The control group and pTPLPV3-inoculated tea seedlings did not show any silencing phenotype ( Figure 5 Compared with the control, the expression of CsPDS gene decreased by about 50% after inoculation of tea seedlings with pTPLPV3-CPCsPDS300F ( Figure 6 ).

Claims

1. A tea tree reticular spot virus infectious cloning vector, characterized in that: Specifically, it is obtained through the following steps: (1) Based on the TPLPV genome information, the cDNA sequences corresponding to the three components of TPLPV were artificially synthesized, namely TPLPV1, TPLPV2 and TPLPV3. When synthesizing TPLPV3, SacI and BamHI restriction sites were added before the stop codon of its first ORF, the mobile protein, and SpeI and NcoI restriction sites were added before the stop codon of its second ORF, the coat protein; (2) TPLPV1, TPLPV2, and TPLPV3 were constructed into the modified pCAMBIA1300 by homologous recombination to obtain pTPLPV1, pTPLPV2, and pTPLPV3, respectively; (3) GFP sequences were incorporated into the 3' end of the MP and CP of pTPLPV3 to further evaluate the expression efficiency of the vector used for TPLPV; (4) Using the Agrobacterium-mediated injection inoculation method, pTPLPV1, pTPLPV2, and pTPLPV3 recombinant viral plasmids were mixed in equal proportions, or pTPLPV3 viral recombinant plasmid was used alone to infect tobacco plants at the 3-5 leaf stage; The sequences of TPLPV1, TPLPV2 and TPLPV3 are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; The modified pCAMBIA1300 plasmid contains a 35S promoter and a NOS terminator. The sequences of the 35S promoter and the NOS terminator are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

2. Use of the tea tree reticulate spot virus infectious cloning vector as claimed in claim 1 in the study of tea tree reticulate spot virus protein function, analysis of viral gene pathogenicity mechanism, and construction and modification of silencing vectors.

3. An Agrobacterium strain carrying an infectious clone of Tea Web Spot Virus, characterized by: The tea tree web spot virus infectious cloning vector according to claim 1 is introduced into the Agrobacterium strain GV3101 through the freeze-thaw method to obtain the vector.

4. Use of the Agrobacterium strain according to claim 3 in the study of the protein function of tea leaf spot virus, analysis of the pathogenic mechanism of viral genes, and construction and modification of silencing vectors.

5. A tea tree reticular spot virus gene silencing vector, characterized in that: These include pTPLPV1, pTPLPV2, and pTPLPV3-VIGS; The pTPLPV3-VIGS is obtained by constructing the target gene fragment to be silenced onto the pTPLPV3 linearized vector after NcoI digestion through homologous recombination method; The pTPLPV1, pTPLPV2 and pTPLPV3 are prepared by constructing the cDNA sequence of the tea tree web spot virus TPLPV into the modified pCAMBIA1300 plasmid; The cDNA sequence of the tea tree web spot virus TPLPV includes TPLPV1, TPLPV2 and TPLPV3, and the sequences of TPLPV1, TPLPV2 and TPLPV3 are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; The modified pCAMBIA1300 plasmid contains a 35S promoter and a NOS terminator, and the sequences of the 35S promoter and the NOS terminator are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; SpeI and NcoI restriction sites are added before the termination codon of the second ORF of the pTPLPV3, namely the coat protein.

6. The use of a tea tree web spot virus gene silencing vector as described in claim 5 in gene silencing of tobacco or tea plants, wherein pTPLPV3-VIGS is inoculated alone or mixed with pTPLPV1 and pTPLPV2 chains by Agrobacterium-mediated method, and the NbPDS gene of the model plant tobacco can be silenced and an albino silenced phenotype can be produced. pTPLPV3-VIGS can be infected alone and the CsPDS gene of tea plants can be silenced and an albino silenced phenotype can be produced.

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