A gene silencing vector induced by Trichosanthes mottle mosaic virus and its construction method and application

Abstract

The invention discloses a gene silencing vector induced by Trichosanthes mottle mosaic virus, a construction method and an application thereof, wherein a Trichosanthes mottle mosaic virus infectious cloning vector is introduced into a single multiple cloning site to obtain a pCB301-TrMMV-MCS gene silencing vector skeleton, and endogenous gene fragments of Nicotiana benthamiana and Cucurbitaceae plants are cloned by an RT-PCR method, and inserted into the multiple cloning site of pCB301-TrMMV-MCS to obtain a gene silencing vector, which induces good silencing of Nicotiana benthamiana endogenous genes by a method of Agrobacterium injection inoculation and friction transfer, and the silencing phenotype can last to the calyx. The gene silencing vector can induce gene silencing of Cucurbitaceae plants including zucchini, cucumber and melon; it exhibits a good silencing effect in melon, and can cause an obvious gene silencing phenotype of melon flowers. The gene silencing vector of the invention provides an effective tool for the study of gene functions of plants such as Cucurbitaceae.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a gene silencing vector induced by Trichosanthes mottle mosaic virus (TrMMV), a construction method and an application thereof. Background Art

[0002] Cucurbitaceae contains many important economic vegetables and medicinal plants. Although the genomes of many species have been sequenced, the difficulty and long period of genetic transformation of Cucurbitaceae plants have limited the study of their gene functions and the selection and promotion of high-yield and high-quality varieties. Virus-induced gene silencing (VIGS) technology is a technology developed based on the plant's defense mechanism against viruses. It usually refers to the gene silencing of plants after being infected by viruses carrying part of the target fragment, resulting in phenotypic variation, and then analyzing the function of the target gene based on the phenotypic change. VIGS technology is widely used to study the function of plant genes because of its unique advantages such as rapidity, low cost, overcoming gene function redundancy, and being unaffected by genotype. Therefore, the development of efficient and convenient VIGS vectors will promote the study of gene functions in Cucurbitaceae plants, thereby promoting the selection and breeding of their excellent varieties.

[0003] At present, the VIGS vectors reported to be applicable to Cucurbitaceae plants are very limited, including only Apple latent spherical virus (ALSV), Tobacco ringspot virus (TRSV), Tobacco rattle virus (TRV), Cucumber green mottle mosaic virus (CGMMV) and Cucumber fruit mottle mosaic virus (CFMMV), and the existing VIGS systems have not been widely used in the gene function of Cucurbitaceae plants. Different virus characteristics will lead to different application scopes and silencing efficiencies of gene silencing vectors. Therefore, selecting suitable viruses and carrying out efficient transformation is the development trend of VIGS vectors. Summary of the invention

[0004] The invention aims to provide a gene silencing vector induced by Trichosanthes mottle mosaic virus, a construction method and an application thereof, so as to ensure its infectivity to a host and to carry exogenous plant gene fragments for stable expression, and finally to achieve silencing of endogenous plant genes.

[0005] Specifically, the technical solution adopted by the present invention is as follows:

[0006] The invention discloses a method for constructing a gene silencing vector induced by Trichosanthes mottle mosaic virus, which specifically comprises the following steps: modifying a Trichosanthes mottle mosaic virus infectious cloning vector, introducing a single multiple cloning site, obtaining a pCB301-TrMMV-MCS gene silencing vector skeleton, cloning an endogenous gene fragment of Nicotiana benthamiana or a cucurbitaceae plant by an RT-PCR method, and inserting the fragment into the multiple cloning site of pCB301-TrMMV-MCS to obtain the gene silencing vector.

[0007] Furthermore, the Nicotiana benthamiana endogenous gene fragment is the endogenous gene NbSu.

[0008] Furthermore, the endogenous gene fragment of the Cucurbitaceae plant is CuPDS90, CuPDS150, CuPDS213 or CuPDS400.

[0009] Further, the following steps are included:

[0010] (1) A single multiple cloning site was inserted into the infectious cloning vector of Trichosanthes mottle mosaic virus by molecular means to obtain the pCB301-TrMMV-MCS vector backbone;

[0011] (2) According to the endogenous gene sequence of Nicotiana benthamiana in Genbank, primers were designed and the endogenous gene NbSu of Nicotiana benthamiana was amplified by RT-PCR. After being inserted into the multiple cloning site of pCB301-TrMMV-MCS, the Nicotiana benthamiana gene silencing vector pCB301-TrMMV-NbSu was obtained;

[0012] Alternatively, according to the conserved sequence of endogenous gene PDS of Cucurbitaceae plants, primers are designed and the endogenous genes CuPDS90, CuPDS150, CuPDS213 or CuPDS400 of Cucurbitaceae plants are amplified by RT-PCR. The endogenous genes are respectively inserted into the pCB301-TrMMV-MCS multiple cloning site to obtain the Cucurbitaceae plant gene silencing vector pCB301-TrMMV-CuPDS90, pCB301-TrMMV-CuPDS150, pCB301-TrMMV-CuPDS213 or pCB301-TrMMV-CuPDS400.

[0013] Wherein: the step (1) specifically includes:

[0014] 1.1) Using pCB301-TrMMV infectious cloning plasmid as a template, CP-TCG-F / CP-TCG-R primers (as shown in SEQ ID No: 1-2) were designed for reverse amplification to obtain gene fragments. The PCR products were treated with DpnI enzyme and then purified by gel recovery. The purified fragments were connected by homologous recombination, transformed, extracted plasmids and sequenced to obtain mutant plasmid pCB301-TrMMV-CP (TCG) in which the start codon of TrMMV genome CP was changed from "ATG" to "ACG".

[0015] 1.2) Design primers TrMMV-VIGS-insert1F / 1R, TrMMV-VIGS-insert2F / 2R and TrMMV-VIGS-insert3F / 3R primer pairs (as shown in SEQ ID No: 3-8) to amplify fragments in three sections (insert1, insert2 and insert3), wherein insert1 and insert2 use pCB301-TrMMV-CP (TCG) as template to obtain a sequence containing 1-5953 nt of TrMMV virus (the fragment contains 78 nt of CP, and the start codon of CP is changed from ATG to ACG), and two restriction sites (ScaI and ClaI) are added to the end AGTACTATCGAT insert3 was amplified using pCB301-TrMMV plasmid as a template to obtain a 5743-6524nt sequence containing TrMMV (CP predicted promoter + CP + 3'UTL), and a sequence with two restriction sites (AscI and BamHI) added to the first end GGCGCGCCGGATCC Insert1, insert2 and insert3 were purified by gel recovery and homologous recombination with the pCB301 plasmid linearized by StuI and SmaI. The correctly recombined pCB301-TrMMV-MCS infectious cloning vector was obtained through positive clone screening, plasmid extraction and sequencing verification.

[0016] In the step (2), primers NbSu215-insertF / R (shown in SEQ ID Nos: 9-10) for amplifying the endogenous Su sequence of Nicotiana benthamiana and primers CuPDS90-insertF / R, CuPDS150-insertF / R, CuPDS213-insertF / R and CuPDS400-insertF / R (shown in SEQ ID Nos: 11-18) for amplifying the endogenous PDS sequences of Cucurbitaceae plants are designed. The sequences are amplified and gel-purified by RT-PCR, and then homologously recombined with the pCB301-TrMMV-MCS vector linearized after ScaI and BamHI digestion. Finally, a TrMMV virus-induced gene silencing vector containing cDNA fragments of endogenous genes of Nicotiana benthamiana and Cucurbitaceae plants is obtained.

[0017] The gene silencing vector induced by the Trichosanthes mottle mosaic virus constructed by the method of the present invention can be used for silencing endogenous genes in Nicotiana benthamiana or Cucurbitaceae plants.

[0018] Among them, the gene silencing vector was transformed into Agrobacterium, and the Agrobacterium containing the pCB301-TrMMV-NbSu vector was infiltrated into Nicotiana benthamiana, that is, the TrMMV-related gene silencing vector plasmid was introduced into the Agrobacterium strain EHA105 by chemical transformation. The EHA105 strain containing the silencing vector was cultured overnight to OD 600 The value was about 1.5. After the cells were collected by centrifugation, an infiltration buffer (containing 10 mM MgCl2, 10 mM MES (pH 5.6), 100 μM acetosyringone, OD 600 =1.0) and injected into 4-week-old Nicotiana benthamiana with a needleless syringe. Two fully expanded new leaves were selected for injection to induce the chlorotic and yellowing silencing phenotype of the endogenous Su gene in Nicotiana benthamiana. The Nicotiana benthamiana leaves inoculated with pCB301-TrMMV-NbSu vector to produce the gene silencing phenotype were further transferred by friction to Nicotiana benthamiana, resulting in a good gene silencing phenotype, and some new leaves showed complete chlorotic and yellowing.

[0019] Among them, Agrobacterium containing pCB301-TrMMV-CuPDS213 vector was inoculated into zucchini, cucumber and melon, that is, the fully expanded cotyledons were impregnated with a needleless syringe; the plants were cultured in a growth room at 25°C with a photoperiod of 16:8h. The phenotype of chlorotic and albino leaves of three Cucurbitaceae plants was induced. Among them, the vectors with different sizes of CuPDS fragments inserted were inoculated into melon, which caused a good gene silencing phenotype in the systemic leaves of melon, and some systemic leaves showed obvious chlorotic and albino. In particular, the pCB301-TrMMV-CuPDS213 vector caused an obvious chlorotic and albino phenotype in melon flowers.

[0020] Compared with the prior art, the outstanding effects of the present invention are:

[0021] (1) Trichosanthes mottle mosaic virus (TrMMV) is a new virus of the genus Tobacco mosaic virus that can infect a variety of cucurbitaceae plants such as Trichosanthes, cucumber, melon, zucchini, watermelon and loofah, as well as Nicotiana benthamiana. Its wide host range may make it a promising candidate for development into an excellent VIGS vector for cucurbitaceae. The present invention utilizes the TrMMV CP subgenomic promoter strategy to drive the expression of plant endogenous genes through the CP promoter while ensuring the infectivity of the virus, thereby achieving silencing of endogenous genes in Nicotiana benthamiana and a variety of cucurbitaceae plants, and obtaining a VIGS tool for application in important economic plants such as cucurbitaceae.

[0022] (2) The present invention efficiently completes the transformation of the TrMMV-VIGS vector by introducing a viral promoter sequence that drives the expression of an exogenous sequence and a multiple cloning restriction site through three-stage homologous recombination, thereby achieving rapid transformation and application of the target vector.

[0023] (3) The present invention successfully achieved systemic infection of host plants by modifying the infectious clone of TrMMV, and reduced pathogenicity, which is suitable for the development of VIGS vectors. Among them, the pCB301-TrMMV-NbSu vector constructed by the present invention achieved good silencing of endogenous genes in Nicotiana benthamiana, and the silencing phenotype can last until the calyx, showing a good and long-lasting silencing effect.

[0024] (4) The VIGS vector provided by the present invention can achieve a good gene silencing effect in melon, achieve complete chlorosis and albinism of systemic leaves, and can cause obvious chlorosis and albinism phenotype of melon flowers. However, there is no VIGS vector applied to Cucurbitaceae in the prior art that shows silencing effect in melon, especially in flowers.

[0025] (5) Based on the method for constructing a gene silencing vector induced by Trichosanthes mottle mosaic virus provided by the present invention, different sequences can be inserted into the multiple cloning site of the vector for the study of other functional genes of Cucurbitaceae crops.

[0026] The gene silencing vector induced by Trichosanthes mottle mosaic virus, its construction method and application described in the present invention are further described below in conjunction with the accompanying drawings and specific examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram for the construction of pCB301-TrMMV-MCS vector

[0028] Figure 2The results of PCR amplification, positive clone screening and restriction enzyme digestion verification of the recombinant fragment of pCB301-TrMMV-MCS. (A) Segmented amplification fragment used for homologous recombination. (B) PCR amplification results of recombinant transformants; 1-15: 15 randomly selected colonies; P: plasmid amplification positive control; N: ddH2O amplification negative control. (C) Plasmid PmeI and SmaI restriction enzyme digestion verification results; 1-4: plasmids extracted from 4 selected positive colonies.

[0029] Figure 3 The pathogenicity of pCB301-TrMMV-MCS and other vectors to N. benthamiana. Among them, (A) the phenotype of N. benthamiana inoculated with pCB301 empty vector for 16 days; (B) the phenotype of N. benthamiana inoculated with pCB301-TrMMV vector for 16 days; (C) the phenotype of N. benthamiana inoculated with pCB301-TrMMV-MCS vector for 16 days. (D) RT-PCR confirmed the systemic infection of N. benthamiana by pCB301-TrMMV-MCS; 1: pCB301 empty vector inoculated plants; 2: pCB301-TrMMV inoculated plants; 3: pCB301-TrMMV-MCS inoculated plants.

[0030] Figure 4 The gene silencing effect of pCB301-TrMMV-NbSu vector in Nicotiana benthamiana. (A) Gene silencing phenotype caused by Agrobacterium pCB301-TrMMV-NbSu inoculation of Nicotiana benthamiana for 35 days. (B) Silencing effect of Agrobacterium pCB301-TrMMV-NbSu infected plants and friction-transferred Nicotiana benthamiana for 18 days. (C) Silencing effect of Agrobacterium pCB301-TrMMV-NbSu infected plants and friction-transferred Nicotiana benthamiana for 50 days.

[0031] Figure 5 In the figure, (A) shows the silencing phenotype (30 dpi) caused by the pCB301-TrMMV-CuPDS213 vector in zucchini, cucumber and melon; (B) shows the qRT-PCR results of endogenous PDS genes in Cucurbitaceae plants.

[0032] Figure 6The silencing effects of different fragment sizes of pCB301-TrMMV-CuPDS on melon. (A) The phenotype of melon inoculated with pCB301-TrMMV-MCS vector; (B) The phenotype of melon inoculated with pCB301-TrMMV-CuPDS90 vector; (C) The phenotype of melon inoculated with pCB301-TrMMV-CuPDS150 vector; (D) The phenotype of melon inoculated with pCB301-TrMMV-CuPDS213 vector; (E) The phenotype of melon inoculated with pCB301-TrMMV-CuPDS400 vector; (F) The qRT-PCR results of the endogenous PDS gene in melon.

[0033] Figure 7 The silencing effect of pCB301-TrMMV-CuPDS213 vector on melon flowers. (A) The phenotype of melon flowers inoculated with pCB301-TrMMV-MCS vector; (B) The albino phenotype of melon flowers caused by pCB301-TrMMV-CuPDS213 vector. DETAILED DESCRIPTION

[0034] Unless otherwise specified, the following examples are based on conventional experimental conditions, and the primer sequences used are shown in Table 1.

[0035] Table 1 Primers constructed based on TrMMV VIGS vector

[0036]

[0037] Example 1 Construction of pCB301-TrMMV-MCS and detection of its infectivity

[0038] 1. Construction of pCB301-TrMMV-MCS

[0039] Using pCB301-TrMMV infectious cloning plasmid as template, CP-TCG-F / CP-TCG-R primers (as shown in SEQ ID No: 1-2) were designed for reverse amplification to obtain gene fragments. The PCR amplification system was: 10 μL 5×TransStart FastPfuBuffer, 4 μL 2.5 mM dNTPs, 1 μL each of upstream and downstream primers (10 μM), 1 μL cDNA template, 1 μL TransStartFastPfu DNA Polymerase, 32 μL ddH2O, and the total reaction system was 50 μL; the amplification reaction conditions were: 95°C for 3 min; 95°C for 30 s, 55°C for 5 min, 72°C for 30 s, 30 cycles; 72°C for 8 min. The PCR product was treated with DpnI enzyme for digestion at 37°C for 30 min, and then purified by gel recovery. The purified fragment was homologously recombined using IIOne Step Cloning Kit (Nanjing Novozyme Biotechnology Co., Ltd.) was used for the reaction, using a 10 μL system, including 2 μL of 5×CEⅡBuffer, 50 ng of purified fragments, 1 μL of ExnaseⅡ, and 10 μL of ddH2O. The recombination reaction conditions were 37°C for 30 min. After transformation, plasmid extraction and sequencing verification of the product, a mutant plasmid pCB301-TrMMV-CP (TCG) in which the start codon of the TrMMV genome CP changed from "ATG" to "ACG" was obtained.

[0040] Based on the subgenomic promoter strategy of tobacco mosaic virus CP, primer pairs TrMMV-VIGS-insert1F / 1R, TrMMV-VIGS-insert2F / 2R and TrMMV-VIGS-insert3F / 3R (as shown in SEQ ID No: 3-8) were designed to amplify fragments in three segments (insert1, insert2 and insert3), wherein insert1 and insert2 used pCB301-TrMMV-CP (TCG) as plasmid templates, and insert3 used pCB301-TrMMV plasmid as template. Through segmented amplification, the sequences of insert1 of TrMMV genome 1-2940nt, 2941-5953nt (the fragment contains 78nt of CP, and the start codon of CP is changed from ATG to ACG, and two restriction sites (ScaI and ClaI) are added at the end) were obtained. AGTACTATCGAT ;) insert2, 5743-6524nt (CP predicted promoter + CP + 3'UTL, and two restriction sites (AscI and BamHI) were added to the first end GGCGCGCCGGATCC ) of insert3, the electrophoresis results are as follows Figure 2 As shown in A, the band sizes were consistent with the expected 2970 bp, 3045 bp and 826 bp. The three fragments were purified by gel recovery and then homologously recombined with the pCB301 plasmid linearized by StuI and SmaI. After E. coli transformation and positive clone screening, 13 out of 15 colonies were determined to be positive ( Figure 2 B). Four positive colonies were selected for culture and plasmid extraction. The plasmid size was confirmed to be consistent with the expected result of two bands of 8068 bp and 3284 bp by PmeI and SmaI digestion. Figure 2 C), and further sequence verification was performed to obtain the recombinant vector pCB301-TrMMV-MCS, in which the CP promoter and the multi-cloning site containing (ScaI, ClaI, AscI and BamHI) were introduced after the TrMMV MP.

[0041] 2. Pathogenicity determination of pCB301-TrMMV-MCS

[0042] The recombinant plasmid pCB301-TrMMV-MCS was transformed into Agrobacterium tumefaciens EH105, and after positive colonies were screened and cultured, the infiltration buffer was prepared and injected into Nicotiana benthamiana. The symptoms of the plants were observed after inoculation. Nicotiana benthamiana ( Figure 3 A) and pCB301-TrMMV inoculated Nicotiana benthamiana ( Figure 3 B) are negative and positive controls. On the 16th day after inoculation with pCB301-TrMMV-MCS, the leaves of Nicotiana benthamiana system showed curling phenotype, which was significantly higher than that of the original pCB301-TrMMV infectious clone ( Figure 3 B), the mutant pCB301-TrMMV-MCS caused milder symptoms in plants ( Figure 3 C). RT-PCR was performed on each sample using TrMMV-specific detection primers. Both pCB301-TrMMV and pCB301-TrMMV-MCS were able to detect the presence of TrMMV ( Figure 3 D), indicating that the constructed pCB301-TrMMV-MCS backbone vector is also pathogenic, but with lower pathogenicity, which is consistent with the characteristics of VIGS vector construction and can be used for further research.

[0043] Example 2 Construction and application of Nicotiana benthamiana silencing vector based on TrMMV

[0044] 1. Construction of Nicotiana benthamiana silencing vector based on TrMMV

[0045] The total RNA of Nicotiana benthamiana was extracted with Trizol, and the cDNA fragment NbSu of the Su gene of Nicotiana benthamiana was amplified by RT-PCR. The primers used were shown in SEQ ID No: 9-10. The PCR amplification system was: 10 μL 5×TransStart FastPfuBuffer, 4 μL 2.5 mM dNTPs, 1 μL each of upstream and downstream primers (10 μM), 1 μL cDNA template, 1 μL TransStartFastPfu DNA Polymerase, 32 μL ddH2O, and the total reaction system was 50 μL; the amplification reaction conditions were: 95°C for 3 min; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 8 min. The pCB301-TrMMV-VIGS-MCS plasmid was double-digested with ScaI and BamHI, and after gel recovery and purification, it was homologously recombined with the purified NbSu215. A 10μL system was used for reaction, including 2μL of 5×CEⅡBuffer, 50ng of purified fragment, 1μL of ExnaseⅡ, and 10μL of ddH2O. The recombination reaction conditions were 37℃30min. Then, E. coli transformation, positive clone screening, plasmid extraction and sequencing verification were performed to obtain the correct pCB301-TrMMV-NbSu vector.

[0046] 2. Application of TrMMV-based Nicotiana benthamiana silencing vector

[0047] The pCB301-TrMMV-NbSu plasmid was transferred into the EHA105 Agrobacterium strain by chemical transformation. 500 ng of plasmid was added to 100 μL of EHA105 Agrobacterium competent medium, flicked to mix, ice bathed for 5 min, treated with liquid nitrogen for 5 min, heat shocked at 37 ° C for 5 min, ice bathed for 2 min, 800 μL of LB recovery medium was added, and cultured at 28 ° C 220 rpm for 2 h. The bacteria were collected by centrifugation and spread on LB solid plates containing kanamycin (50 mg / L) and rifampicin (50 mg / L). After culturing at 28 ° C for 2 days, single clones were picked for NbSu screening, and positive clones were inoculated in LB liquid medium containing kanamycin (50 mg / L) and rifampicin (50 mg / L), and cultured overnight at 28 ° C 220 rpm. When the bacterial solution OD 600 When the OD was about 1.5, the cells were collected by centrifugation and resuspended in infiltration buffer (final concentration of 10 mM MgCl2, 10 mM MES (pH 5.6), 100 μM acetosyringone (Acs)) to adjust the concentration of the bacterial solution to OD 6001.0, and placed in dark place at room temperature for 2-3 hours. Using 4-5 leaf stage Nicotiana benthamiana as test material, 3 fully expanded upper leaves were infiltrated with a 1mL syringe without a needle, and the plants were cultured in a growth room at 25°C with a photoperiod of 16:8h. About 16 days after inoculation, Nicotiana benthamiana inoculated with pCB301-TrMMV-NbSu began to show chlorotic and yellowing phenotypes, and on the 35th day, some leaves showed obvious chlorotic and yellowing ( Figure 4 A). The leaves of Nicotiana benthamiana that produced the silent phenotype after inoculation with pCB301-TrMMV-NbSu were transferred to Nicotiana benthamiana by friction. About 11 days after inoculation, the new leaves of Nicotiana benthamiana showed a chlorotic and yellowing phenotype. Subsequently, the systemic leaves showed a good silent phenotype, and most systemic leaves showed complete chlorosis ( Figure 4 B). Subsequently, a clear chlorosis-yellowing phenotype was also observed in the calyx of P. benthamiana ( Figure 4 C). This indicates that the pCB301-TrMMV-NbSu vector has a good and lasting silencing effect on Nicotiana benthamiana.

[0048] Example 3 Construction and application of TrMMV-based gene silencing vector for Cucurbitaceae plants

[0049] 1. Construction of TrMMV-based Cucurbitaceae Silencing Vector

[0050] Total RNA of cucumber was extracted by Trizol and reverse transcribed to synthesize cDNA. The PDS genes of plants such as cucumber, melon, and zucchini were compared, and the more conservative regions were selected. The cDNA of the representative Cucurbitaceae plant cucumber was used as a template for amplification. CuPDS90-insertF / R, CuPDS150-insertF / R, CuPDS213-insertF / R, and CuPDS400-insertF / R (as shown in SEQ ID No: 11-18) were used for amplification to obtain CuPDS gene fragments with lengths of 90 bp, 150 bp, 213 bp, and 400 bp. The PCR amplification system was: 10 μL 5×TransStart FastPfu Buffer, 4 μL 2.5 mM dNTPs, 1 μL each of upstream and downstream primers (10 μM), 1 μL cDNA template, 1 μL TransStart FastPfu DNA Polymerase, 32μL ddH2O, the total reaction system is 50μL; the amplification reaction conditions are: 95℃3min; 95℃30s, 55℃30s, 72℃30s, 35 cycles; 72℃8min. After gel recovery and purification, the product was homologously recombined with the pCB301-TrMMV-MCS plasmid linearized by ScaI and BamHI enzymes, and a 10μL system reaction was used, including 5×CEⅡBuffer 2μL, 50ng of purified fragment, 1μL of ExnaseⅡ, ddH2O to make up the system to 10μL, and the recombination reaction conditions were 37℃30min. After transformation into E. coli, positive clones were screened and verified by restriction digestion and sequencing, and finally the vectors pCB301-TrMMV-CuPDS90, pCB301-TrMMV-CuPDS150, pCB301-TrMMV-CuPDS213, and pCB301-TrMMV-CuPDS400 with the CuPDS fragment correctly inserted were obtained.

[0051] 2. Application of TrMMV-based silencing vector for Cucurbitaceae plants

[0052] The pCB301-TrMMV-CuPDS213 plasmid was transferred into the EHA105 Agrobacterium strain. After obtaining the Agrobacterium cells, they were resuspended in the infiltration buffer and the concentration of the bacterial solution was adjusted to OD 600 1.0, and placed in dark place at room temperature for 2-3 hours. For Agrobacterium inoculation of Cucurbitaceae plants, fully expanded cotyledons were impregnated with a needleless syringe, and then the plants were placed in a growth room at 25°C for cultivation with a photoperiod of 16:8h. About 30 days after inoculation, all plants of zucchini, cucumber and melon inoculated with pCB301-TrMMV-CuPDS213 induced the typical silent symptom of leaf photobleaching ( Figure 5A). qRT-PCR results showed that the expression of endogenous PDS genes in Cucurbitaceae plants was significantly inhibited ( Figure 5 B).

[0053] The plasmids pCB301-TrMMV-CuPDS90, pCB301-TrMMV-CuPDS150, pCB301-TrMMV-CuPDS213, and pCB301-TrMMV-CuPDS400 were further transferred into the EHA105 Agrobacterium strain, and the melons were infiltrated with Agrobacterium after obtaining the bacterial suspension. The melons were cultured in a growth room at 25°C with a photoperiod of 16:8h. On the 30th day after inoculation, the melon system leaves showed obvious photobleaching phenotype, and some leaves showed complete chlorosis and whitening ( Figure 6 AE). qRT-PCR results showed that the endogenous PDS gene in melon was significantly silenced ( Figure 6 F). Continued observation of melons inoculated with the pCB301-TrMMV-CuPDS213 vector revealed that the flowers in the silenced plants showed obvious photobleaching phenotype ( Figure 7 ), indicating that the pCB301-TrMMV-VIGS vector has good application prospects in melon flowers.

[0054] Based on the method for constructing a gene silencing vector induced by Trichosanthes mottle mosaic virus provided by the present invention, different sequences can be inserted into the multiple cloning site of the vector for the study of other functional genes of Nicotiana benthamiana or Cucurbitaceae crops, and are not limited to the endogenous gene NbSu of Nicotiana benthamiana and the endogenous genes CuPDS90, CuPDS150, CuPDS213 and CuPDS400 of Cucurbitaceae plants.

[0055] The embodiments described above are merely descriptions of preferred implementation 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. A method for constructing a gene silencing vector induced by Trichosanthes mottle mosaic virus, characterized in that: The infectious cloning vector of Trichosanthes mottle mosaic virus was modified, and a single multiple cloning site was introduced to obtain the pCB301-TrMMV-MCS gene silencing vector skeleton. The endogenous gene fragment of Nicotiana benthamiana or Cucurbitaceae plants was cloned by RT-PCR and inserted into the multiple cloning site of pCB301-TrMMV-MCS to obtain the gene silencing vector; the endogenous gene fragment of Nicotiana benthamiana is the endogenous gene Nb ; The endogenous gene fragment of the Cucurbitaceae plant is CuPDS 90. CuPDS 150, CuPDS 213 or CuPDS 400, that is, using cucumber cDNA as a template for amplification, using CuPDS90-insertF / R, CuPDS150-insertF / R, CuPDS213-insertF / R and CuPDS400-insertF / R for amplification, respectively, to obtain CuPDS gene fragments with lengths of 90 bp, 150 bp, 213 bp and 400 bp; the sequences of the CuPDS90-insertF / R, CuPDS150-insertF / R, CuPDS213-insertF / R and CuPDS400-insertF / R are shown in SEQ ID No: 11-18; The following steps are involved: (1) Using molecular methods, a multiple cloning site was inserted into the infectious cloning vector of Trichosanthes mottle mosaic virus to obtain the pCB301-TrMMV-MCS vector backbone; 1.1) Using pCB301-TrMMV infectious cloning plasmid as template, design CP-TCG-F / CP-TCG-R primers as shown in SEQ ID No: 1-2, reverse amplify to obtain gene fragments, and use Dpn The PCR product was treated with I enzyme and then purified by gel recovery. The purified fragment was connected by homologous recombination, transformed, extracted plasmid and sequenced to obtain the mutant plasmid pCB301-TrMMV-CP (TCG) in which the start codon of the TrMMV genome coat protein CP changed from "ATG" to "ACG". 1.2) Design primer pairs TrMMV-VIGS-insert1F / 1R, TrMMV-VIGS-insert2F / 2R and TrMMV-VIGS-insert3F / 3R, as shown in SEQ ID No: 3-8, and amplify fragments in three segments, insert1, insert2 and insert3, wherein insert1 and insert2 use pCB301-TrMMV-CP (TCG) as templates to obtain 1-5953 nt containing the TrMMV genome, wherein the fragment contains 78 nt of CP, and the start codon of CP is changed from ATG to ACG, and two restriction sites are added to the end Sca I and Cla Sequence of I AGTACTATCGAT ; insert3 was amplified using pCB301-TrMMV plasmid as template to obtain the 5743-6524 nt sequence of TrMMV, CP predicted promoter + CP + 3'UTL, and two restriction sites were added at the first end Asc I and Bam Sequence of HI GGCGCGCCGGATCC ; Insert1, insert2 and insert3 were purified by gel recovery and Stu I and Sma I digested the linearized pCB301 plasmid for homologous recombination and ligation, and the pCB301-TrMMV-MCS infectious cloning vector was obtained through positive clone screening, plasmid extraction and sequencing verification. (2) Based on the endogenous gene sequence of Nicotiana benthamiana in Genbank, primers were designed and the endogenous gene of Nicotiana benthamiana was amplified by RT-PCR Nb , after inserting into the pCB301-TrMMV-MCS multiple cloning site, the Nicotiana benthamiana gene silencing vector pCB301-TrMMV-NbSu was obtained; Alternatively, based on the cucumber endogenous gene PDS The conserved sequences of Cucurbitaceae plants were used to design primers and amplify endogenous genes of Cucurbitaceae plants by RT-PCR. CuPDS 90. CuPDS 150, CuPDS 213 or CuPDS 400, and the endogenous genes were inserted into the pCB301-TrMMV-MCS multiple cloning site to obtain the Cucurbitaceae gene silencing vector pCB301-TrMMV-CuPDS90, pCB301-TrMMV-CuPDS150, pCB301-TrMMV-CuPDS213 or pCB301-TrMMV-CuPDS400; Designing the endogenous source of Nicotiana benthamiana Su Sequence amplification primer NbSu215-insertF / R, as shown in SEQ ID No: 9-10; or, each endogenous PDS Sequence amplification primers CuPDS90-insertF / R, CuPDS150-insertF / R, CuPDS213-insertF / R or CuPDS400-insertF / R, as shown in SEQ ID Nos: 11-18; The above sequence was amplified by RT-PCR and purified by gel recovery. Sca I and Bam The pCB301-TrMMV-MCS vector linearized after HI digestion was homologously recombined and connected to finally obtain the TrMMV virus-induced gene silencing vector containing the cDNA fragment of the endogenous gene of Nicotiana benthamiana or Cucurbitaceae plants.

2. The gene silencing vector induced by Trichosanthes mottle mosaic virus obtained by the construction method of claim 1.

3. Use of the gene silencing vector induced by the Trichosanthes mottle mosaic virus according to claim 2 in silencing endogenous genes in Nicotiana benthamiana or Cucurbitaceae plants.

4. Use of the gene silencing vector induced by Trichosanthes mottle mosaic virus in silencing endogenous genes in Nicotiana benthamiana or Cucurbitaceae plants according to claim 3, characterized in that: The gene silencing vector was transformed into Agrobacterium, and the Agrobacterium containing the pCB301-TrMMV-NbSu vector was infiltrated into Nicotiana benthamiana to induce Nicotiana benthamiana to produce endogenous Su The gene silencing phenotype of chlorosis and yellowing was further obtained by friction transfer of Nicotiana benthamiana leaves inoculated with pCB301-TrMMV-NbSu vector to Nicotiana benthamiana, resulting in a good gene silencing phenotype.

5. Use of the gene silencing vector induced by Trichosanthes mottle mosaic virus according to claim 3 in silencing endogenous genes in Nicotiana benthamiana or Cucurbitaceae plants, characterized in that: Agrobacterium containing the pCB301-TrMMV-CuPDS213 vector was inoculated into zucchini, cucumber and melon to induce the chlorotic and whitening phenotype of the leaves of the three Cucurbitaceae plants; CuPDS The vector with the fragment inserted was inoculated into melon, which caused a good gene silencing phenotype in the systemic leaves of melon.

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