A method of virus-induced silencing of a haloxylon gene

Abstract

The present application relates to the technical field of genetic engineering, and in particular to a method for virus-induced silencing of a haloxylon ammodendron gene, The present application provides a method for virus-induced silencing of a haloxylon ammodendron gene, comprising the steps of construction of a VIGS vector, transformation of agrobacterium, preparation of a bacterial solution for transfection, and virus homogenate infection of haloxylon ammodendron plants. The virus vector modified from tobacco etch virus is used to establish a haloxylon ammodendron gene silencing system, and the virus homogenate-mediated gene silencing is faster and more efficient, and the haloxylon ammodendron HaCLA1 gene expression is significantly reduced. The present application first applies the VIGS gene silencing technology to haloxylon ammodendron, and establishes an efficient haloxylon ammodendron gene silencing system, which can efficiently silence the target gene of a plant in a short time, is simple and efficient to operate, has a short experimental period and low cost. Meanwhile, HaCLA1 the gene can be used as a reporter gene for haloxylon ammodendron gene silencing, and the constructed vector pTRV2- HaCLA1 can be used as a positive vector for haloxylon ammodendron gene silencing.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method for silencing the Haloxylon ammodendron gene induced by a virus. Background Technology

[0002] Haloxylon ammodendron (CA Mey.) Bunge is a xerophytic shrub or small tree belonging to the genus Haloxylon in the family Amaranthaceae, growing to a height of 1–9 meters. It is also known as salt tree, saxaul tree, and saxaul firewood. Haloxylon is one of the most widely distributed desert plants in arid regions, exhibiting strong tolerance to high temperatures, drought, salinity, and poor soil conditions. It demonstrates exceptional adaptability to harsh desert environments, making it an ideal material for studying plant stress resistance mechanisms. It is also the tree species with the largest area of ​​sand-fixing afforestation in arid areas, earning it the title of "desert guardian." Haloxylon wood is hard and makes excellent fuelwood. Its current-year assimilative branches are high in crude protein and rich in nutrients, providing excellent fodder for camels, sheep, and other livestock. The valuable traditional Chinese medicine Cistanche deserticola, known as "desert ginseng," parasitizes its roots. Therefore, Haloxylon has significant ecological, feed, and economic value.

[0003] Among existing genomic research methods, virus-induced gene silencing (VIGS) is a relatively mature transgenic technology widely used in monocotyledonous and dicotyledonous plants, such as tomatoes, tobacco, cotton, peppers, Arabidopsis thaliana, potatoes, corn, wheat, and petunias. Silencing of target genes can be achieved in contemporary plants through inoculation with viral vectors. VIGS was initially used to explain the plant's defense mechanisms after viral infection, a phenomenon prevalent in plants. VIGS utilizes the plant's inherent RNA interference and viral immune response mechanisms.

[0004] There are very few reports in the literature regarding the establishment of stable regeneration and genetic transformation systems for Haloxylon ammodendron. It is quite difficult to study the function of Haloxylon ammodendron genes using T-DNA insertion mutation, RNA interference (RNAi), or CRISPR / Cas9 systems. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for virus-induced silencing of Haloxylon ammodendron genes, addressing the shortcomings of the prior art. The method involves constructing a VIGS vector, transforming with Agrobacterium tumefaciens, preparing transfection bacterial solution and virus homogenate, and infecting Haloxylon ammodendron plants. Using tobacco brittle virus (TRV) as the viral vector, a Haloxylon ammodendron silencing system is established. The virus homogenate-mediated gene silencing is faster and more efficient. After silencing, the expression level of the Haloxylon ammodendron HaCLA1 gene is significantly reduced. This method can efficiently silence the target gene in plants in a short time. It is simple to operate, efficient, has a short experimental cycle, and is low in cost.

[0006] This invention provides a method for virus-induced silencing of the Haloxylon ammodendron gene, comprising the following steps:

[0007] Construction of S1 and VIGS vectors:

[0008] S11. Extract total RNA from Haloxylon ammodendron and reverse transcribe it into cDNA. Use Haloxylon ammodendron cDNA as a template to amplify the HaCLA1 fragment containing the nucleotide sequence shown in SEQ ID NO: 1.

[0009] S12, double enzyme digestion to prepare pTRV2 linearized vector;

[0010] S13. The target fragment HaCLA1 and the pTRV2 linearized vector were recovered using a DNA gel recovery kit.

[0011] S14, recombine the target fragment HaCLA1 and pTRV2 linearized vector to obtain the recombination product and link it to the recombination reaction solution;

[0012] S15. The recombinant product reaction solution was transformed into DH5α Escherichia coli competent cells to obtain the recombinant vector pTRV2-HaCLA1.

[0013] S2. Recombinant vector transformation of Agrobacterium: pTRV1, pTRV2, and pTRV2-HaCLA1 plasmids were extracted and transformed into thawed GV3101 Agrobacterium competent cells, respectively. Positive clones were screened to obtain GV3101 Agrobacterium strains with pTRV1, pTRV2, and pTRV2-HaCLA1.

[0014] S3, Infected Haloxylon ammodendron:

[0015] S31. Prepare bacterial suspension for transfection;

[0016] S32. Using tobacco to amplify virus homogenate to infect Haloxylon ammodendron.

[0017] According to the method for silencing the Haloxylon ammodendron gene induced by a virus provided by the present invention, the double digestion system described in S12 is as follows: 5 μL of 10×FastDigest buffer, 1 μL each of EcoRI and KpnI, 1 μL of pTRV2 empty vector, and the volume is supplemented with sterile water to 20 μL. The digestion conditions are 37℃ for 30 min.

[0018] According to the method for virus-induced silencing of the Haloxylon ammodendron gene provided by the present invention, the recombinant reaction system in S14 consists of 10 μL: 5 μL of 2×Uniclone Seamless Cloning Mix, 2 μL of linearized vector pTRV2, and 3 μL of HaCLA1, and reacted at 50 °C for 30 min.

[0019] According to the method for virus-induced silencing of the *Haloxylon ammodendron* gene provided by the present invention, the recombinant product described in S15 is transformed by *E. coli* competent cells DH5α as follows: Take *E. coli* competent cells DH5α, add 10 μL of the ligation recombinant reaction solution prepared in S15, gently mix, and place it on ice for 30 min. After standing, place it in a 42℃ metal bath for 45 s and immediately place it on ice for 2 min. Then add 700 μL of sterilized antibiotic-free LB liquid medium, and then place it in a 37℃ incubator for 1 h at 220 rpm. Then take 100 μL and spread it on LB solid medium resistant to 50 μg / mL kanamycin sulfate, and incubate overnight in a 37℃ incubator.

[0020] According to the method for silencing the Haloxylon ammodendron gene induced by a virus provided by the present invention, the preparation of the transfection bacterial solution in step S31 includes the following steps:

[0021] S311, Activation: The GV3101 Agrobacterium strains pTRV1, pTRV2 and pTRV2-HaCLA1 prepared in S2 were streaked on YEB solid medium to obtain activated Agrobacterium;

[0022] S312, Small-scale shaking strain: Select a single clone of activated Agrobacterium and incubate it overnight at 28°C and 180 rpm in a shaker.

[0023] S313, Large-scale culture: Transfer 100 μL of Agrobacterium cultured in S312 to 10 mL of YEB liquid medium and incubate at 28°C and 180 rpm until OD. 600 =0.7~1.2;

[0024] S314. Treatment of bacterial strains: Centrifuge the product prepared in S313 at 5000 rpm for 10 min, discard the supernatant to enrich the colonies, and resuspend the colonies in resuspending buffer to OD. 600 =0.6~0.8, stand at room temperature for 2~3 h, mix pTRV1 with pTRV2 and pTRV2-HaCLA1 in equal volumes at a ratio of 1:1 to obtain the bacterial solution for transfection.

[0025] According to the method for silencing the Haloxylon ammodendron gene induced by a virus provided by the present invention, 50 μg / mL of rifampicin and 50 μg / mL of kanamycin sulfate are added to the YEB solid culture medium described in S311 and the YEB liquid culture medium described in S312 and S313.

[0026] According to the method for virus-induced silencing of the Haloxylon ammodendron gene provided by the present invention, the resuspension in S314 is obtained by uniformly mixing MS liquid culture medium, 10 mmol / L MgCl2, 10 mmol / L MES and 150 μmol / L AS.

[0027] According to the method for virus-induced silencing of the Haloxylon ammodendron gene provided by the present invention, the method of infecting Haloxylon ammodendron with tobacco amplified virus homogenate as described in S32 is as follows: The transfection bacterial solution prepared in S31 is slowly injected through a wound on a tobacco leaf. After culturing for 7 days, 1 g of tobacco leaves are weighed, the leaves are chopped and placed in a sterile mortar, and then 1 mL of 0.2 mmol / L sodium phosphate buffer with a pH of 7.2 is added. The tobacco leaves are ground into a homogenate in the mortar, and after filtration, the virus solution is obtained. The virus solution is diluted with sterile water at a ratio of 1:40 to prepare a virus infection solution. Four-week-old Haloxylon ammodendron plants are immersed in the virus infection solution and co-cultured in the dark for 48 h at a temperature maintained at 22–24 °C. Afterwards, they are transferred to an incubator to observe the phenotype of the Haloxylon ammodendron plants.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] This invention provides a method for virus-induced gene silencing in Haloxylon ammodendron, comprising the steps of constructing a VIGS vector, Agrobacterium transformation, and preparing transfection bacterial suspension and virus homogenate for infection of Haloxylon ammodendron plants. In this invention, tobacco brittle virus is used as the viral vector to establish a Haloxylon ammodendron silencing system. Results demonstrate that both transfection bacterial suspension and virus homogenate infection of Haloxylon ammodendron plants can induce a gene silencing phenotype, but virus homogenate-mediated gene silencing is faster and more efficient. Simultaneously, qPCR detection of the Haloxylon ammodendron HaCLA1 gene expression level showed a significant decrease in HaCLA1 gene expression after silencing compared to the control group. This invention is the first to practically apply VIGS gene silencing technology in Haloxylon ammodendron, establishing a highly efficient gene silencing system that can efficiently silence target genes in plants within a short time. The method is simple, efficient, has a short experimental cycle, and is low in cost. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 This is a pTRV2-HaCLA1 plasmid map;

[0032] Figure 2This is an agarose gel electrophoresis image of the cloned HaCLA1 gene using PCR.

[0033] Figure 3 This is a flowchart of VIGS mediated by the saxaul TRV virus;

[0034] Figure 4 These are phenotypic diagrams of Haloxylon ammodendron control plants and plants after the HaCLA1 gene was silenced.

[0035] Figure 5 These are phenotypic diagrams of assimilative branches in Haloxylon ammodendron control plants and plants after the HaCLA1 gene was silenced.

[0036] Figure 6 The method involves qPCR to detect changes in the expression level of the HaCLA1 gene in control and silent strains of Haloxylon ammodendron. Detailed Implementation

[0037] Example 1

[0038] The planting method of the raw material Haloxylon ammodendron used in this embodiment is as follows: Haloxylon ammodendron seeds were collected from Turpan City, Xinjiang Uygur Autonomous Region. Plump seeds with similar shape and size were selected, disinfected by soaking in 75% alcohol for 60 seconds, thoroughly mixed by vortexing, and then filtered. The seeds were rinsed, shaken, and filtered three times with sterile water. They were then soaked and shaken in sodium hypochlorite stock solution, allowed to stand for 13 minutes, and then filtered. The seeds were rinsed, shaken, and filtered six times with sterile water. The clean Haloxylon ammodendron seeds were then inoculated into sterile 250 mL tissue culture bottles on MS solid medium, with the seeds kept at a constant distance. The bottles were then placed in a light incubator with a culture temperature of 28℃, a relative humidity of 60-65%, a light condition of 8h / 16h, and a light intensity of 5000 LX to obtain Haloxylon ammodendron assimilating branches.

[0039] Extraction and detection of total RNA from Haloxylon ammodendron: Haloxylon ammodendron assimilating branches were used as raw materials. After being flash-frozen in liquid nitrogen, the branches were thoroughly ground and RNA was extracted using an RNA extraction kit (Sangon Biotech (Shanghai) Co., Ltd., B518661). The RNA was then reverse transcribed into cDNA using a reverse transcription kit (Applied Biological Materials Inc. (ABM), G592).

[0040] This embodiment provides a method for virus-induced silencing of the Haloxylon ammodendron gene, the specific steps of which are as follows:

[0041] S1 and VIGS carrier construction

[0042] S11. Based on the HaCLA1 (cloroplastos alterados 1 gene) gene sequence, a recombinant vector pTRV2-HaCLA1 was constructed with upstream and downstream pTRV2 vector sequences and EcoRI and KpnI restriction sites, respectively. To reduce the introduction of amplification mutations, high-fidelity polymerase 2×PhantaMax Master Mix (Dye Plus) (Vazyme #P525) was used for amplification.

[0043] The nucleotide sequence of HaCLA1 is shown in SEQ ID NO: 1, and its primers are pTRV2-HaCLA1-F and pTRV2-HaCLA1-R, respectively. The nucleotide sequence of primer pTRV2-HaCLA1-F is shown in SEQ ID NO: 2, and the nucleotide sequence of primer pTRV2-HaCLA1-R is shown in SEQ ID NO: 3.

[0044] Preparation of S12 and pTRV2 linearized vectors

[0045] Linearized vectors were prepared by enzyme digestion. Double enzyme digestion was used to completely linearize the vectors and reduce the transformation background. The extracted plasmid pTRV2 was double-digested with Thermo Scientific FastDigest EcoRI and KpnI, respectively.

[0046] EcoRI and KpnI double digestion system: 5 μL of 10×FastDigest buffer, 1 μL each of EcoRI and KpnI, 1 μL of pTRV2 empty vector, and add sterile water to make up to 20 μL. The digestion conditions are 37℃ for 30 min.

[0047] S13, Glue Recycling Target Fragments and Carriers

[0048] Electrophoresis was performed on the target fragment HaCLA1 and the pTRV2 vector, respectively. The gels containing the target fragment HaCLA1 and the linearized vector pTRV2 were excised, and the target fragment and the linearized vector were purified according to the DNA gel extraction kit (MegG).

[0049] Figure 2 The image shows agarose gel electrophoresis result of PCR cloning of the HaCLA1 gene. In the image, M represents the 2K DNA Marker and 1 represents HaCLA1.

[0050] S14, Recombination reaction

[0051] The recombination reaction was carried out according to the Uniclone One Step Seamless Cloning Kit (Jinsha) in a 10 μL system: 5 μL of 2×Uniclone Seamless Cloning Mix, 2 μL of linearized vector pTRV2, and 3 μL of HaCLA1. The reaction was carried out at 50 °C for 30 min.

[0052] S15, Transformation of Recombinant Products

[0053] Remove E. coli competent cells DH5α from the -80℃ freezer and place them on ice. When the E. coli thaws, add 10 μL of ligation and recombination reaction solution, gently swirl and mix, and then place on ice for 30 min.

[0054] After settling, the centrifuge tubes were placed in a 42°C metal bath and reacted for 45 seconds. Immediately afterward, they were placed on ice for 2 minutes. Then, the centrifuge tubes were transferred to a clean bench and 700 μL of sterilized antibiotic-free LB liquid medium was added to the centrifuge tubes. The centrifuge tubes were then placed in a 37°C incubator and thawed at 220 rpm for 1 hour. 100 μL of the thawed medium was then spread onto LB solid medium resistant to kanamycin sulfate (50 μg / mL) and incubated overnight at 37°C.

[0055] S16, Gene Fragment Verification

[0056] Single colonies were picked from overnight LB solid medium containing kanamycin resistance and subjected to bacterial PCR verification. After confirming that the band size was correct, the colonies were sequenced at Shanghai Sangon Biotech Co., Ltd. to obtain the recombinant vector pTRV2-HaCLA1.

[0057] S2, recombinant vector transformed Agrobacterium

[0058] pTRV1, pTRV2, and pTRV2-HaCLA1 plasmids were extracted and transformed into thawed GV3101 Agrobacterium competent cells. Positive clones were screened. According to the instructions of Sangon GV3101 Agrobacterium competent cells, single clones of Agrobacterium growing on the culture medium were picked. Positive single clones were identified by bacterial PCR. After preservation, the cells were stored at -80℃ for later use.

[0059] S3, Infected Haloxylon ammodendron

[0060] S31. Preparation of bacterial solution for transfection and inoculation of Haloxylon ammodendron

[0061] ① Activation: The previously obtained Agrobacterium tumefaciens strains pTRV1, pTRV2, and pTRV2-HaCLA1 were streaked on YEB solid medium;

[0062] YEB solid medium contains rifampicin at a concentration of 50 μg / mL and kanamycin sulfate at a concentration of 50 μg / mL. The preparation method of YEB solid medium is as follows: Measure 31.24 g of YEB medium, add deionized water to make up to 1000 mL, stir well, sterilize at 121℃ for 20 min, cool the medium to about 55℃, then add 1 mL of rifampicin at a concentration of 50 mg / mL and 0.5 mL of kanamycin at a concentration of 100 mg / mL, pour into a culture dish, and store in a refrigerator at 4℃.

[0063] ② Small-scale shaking culture: Select a single Agrobacterium colony and culture it in 1 mL of YEB liquid medium (50 μg / mL rifampicin + 50 μg / mL kanamycin sulfate) overnight at 28℃ and 180 rpm.

[0064] ③ Large-scale culture: Transfer 100 μL of Agrobacterium culture to 10 mL of YEB liquid medium (rifampicin 50 μg / mL + kanamycin sulfate 50 μg / mL), and incubate at 28°C and 180 rpm until OD. 600 =0.7~1.2;

[0065] ④ Processing the bacterial culture: Collect the bacteria, centrifuge at 5000 rpm for 10 min, discard the supernatant, enrich the colonies, and resuspend the enriched colonies in the prepared resuspension solution to OD. 600 =0.6~0.8, stand at room temperature for 2~3 h, mix pTRV1 with pTRV2 and pTRV2-HaCLA1 in equal volumes at a ratio of 1:1 to obtain the bacterial solution for transfection;

[0066] Resuspension (100 mL): Take 100 μL of 150 mM acetylsalicylic acid solution for sterilization, 2 mL of 10 mM MgCl2 solution, and 2 mL of 10 mM MES solution, and add 1×MS liquid culture medium to make up to 100 mL.

[0067] ⑤ Immerse four-week-old Haloxylon ammodendron plants in transfection bacterial solution and co-culture in the dark for 48 hours at a temperature of 22-24℃. Then transfer them to an incubator to observe the phenotype of Haloxylon ammodendron plants.

[0068] S32. Using tobacco-amplified virus homogenate to infect Haloxylon ammodendron

[0069] Using a syringe, the transfection bacterial solution from step S31 was slowly injected through a wound on a tobacco leaf, and cultured for another 7 days. 1 g of tobacco leaf containing virus particles, after being injected with Agrobacterium resuspension and cultured for 7 days, was weighed, chopped, and placed in a sterile mortar. 1 mL of 0.2 M sodium phosphate buffer (PB) at pH 7.2 was added, and the tobacco leaf was ground into a homogenate in the mortar. To remove large particles, the homogenate was filtered through four layers of gauze into a beaker. The filtered liquid was dispensed into 2 mL centrifuge tubes and stored at -20°C for later use. The prepared virus solution was diluted with sterile water at a ratio of 1:40 to prepare a virus infection solution. Four-week-old Haloxylon ammodendron plants were immersed in the virus infection solution and co-cultured in the dark for 48 hours at a temperature maintained at 22–24°C. Afterward, the plants were transferred to an incubator to observe their phenotype.

[0070] Gene expression detection:

[0071] Continuous observation of the whitening phenomenon of pTRV2-HaCLA1 injected Haloxylon ammodendron assimilation branches. The appearance of whitening phenomenon indicates that the expression level of the HaCLA1 gene in Haloxylon ammodendron has been successfully reduced in Haloxylon ammodendron. Haloxylon ammodendron cannot synthesize chlorophyll normally, which marks the successful construction of the silencing system.

[0072] Once the pTRV2-HaCLA1-injected Haloxylon ammodendron exhibits albinoing, pTRV2-HaCLA1 and pTRV2 empty-vectored Haloxylon ammodendron assimilation branches are harvested, rapidly frozen and ground in liquid nitrogen, and RNA is extracted using an RNA extraction kit (Sangon Biotech (Shanghai) Co., Ltd., B518661). The RNA is then reverse transcribed into cDNA using a reverse transcription kit (Applied Biological Materials Inc. (ABM), G592), and the expression level of the HaCLA1 gene is detected by real-time quantitative fluorescence detection.

[0073] The quantitative real-time PCR reaction system is as follows: Each 20 μL reaction system contains 10 μL of BlasTaq™ 2× qPCRMasterMix, 0.5 μL each of forward and reverse primers, 2 μL of template, and is supplemented with sterile water to a volume of 20 μL. The internal reference gene is 18S rRNA. The nucleotide sequence of primer 18S rRNA-F is shown in SEQ ID NO: 4, and the nucleotide sequence of primer 18S rRNA-R is shown in SEQ ID NO: 5. The nucleotide sequence of primer qPCR-HaCLA1-F is shown in SEQ ID NO: 6, and the nucleotide sequence of primer qPCR-HaCLA-R is shown in SEQ ID NO: 7.

[0074] The quantitative PCR reaction program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 60℃ annealing and extension for 60 s, for 40 cycles.

[0075] Real-time quantitative PCR data analysis methods

[0076] The relative expression levels of the target gene in this experiment were measured using a 2-1 -△△CT The method is used to analyze the relative expression level of the target gene, and the formula for calculation is as follows:

[0077] △CT 目的基因 =CT 目的基因 -CT 内参基因

[0078] △CT 对照目的基因 =CT 对照目的基因 -CT 对照内参基因

[0079] △△CT=△CT 目的基因 -△CT 对照目的基因

[0080] The relative expression level of the target gene = 2 －△△CT =2 －（△CT目的基因－△CT对照目的基因）

[0081] Figure 4 The images show the phenotypic diagrams of Haloxylon ammodendron control plants and plants after the HaCLA1 gene was silenced. The left side shows the control plants, and the right side shows the albino plants after the HaCLA1 gene was silenced. Figure 5 The images show the phenotypic diagrams of assimilation branches of Haloxylon ammodendron control plants and plants after the HaCLA1 gene was silenced. The left side shows the assimilation branches of the control plants, and the right side shows the assimilation branches of the albino plants after the HaCLA1 gene was silenced.

[0082] Figure 6 qPCR was used to detect changes in the expression level of the HaCLA1 gene in control and silenced strains of Haloxylon ammodendron. It was found that the expression level of the HaCLA1 gene was significantly reduced after silencing.

[0083] In this embodiment, a gene silencing system for Haloxylon ammodendron was established using tobacco brittle virus as the viral vector. Results demonstrated that both transfection with bacterial suspension and virus homogenate in Haloxylon ammodendron plants resulted in gene silencing phenotypes. However, virus homogenate-mediated gene silencing was faster and more efficient. Simultaneously, qPCR analysis of the HaCLA1 gene expression level in Haloxylon ammodendron showed a significant decrease in HaCLA1 gene expression after silencing compared to the control group. This embodiment marks the first practical application of VIGS gene silencing technology in Haloxylon ammodendron, establishing a highly efficient gene silencing system that can effectively silence target genes in plants within a short time. The system is simple, efficient, has a short experimental cycle, and is low in cost.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method of virus-induced silencing of a Gossypium raimondii gene, characterized in that, The method comprises the following steps: S1, VIGS vector construction: S11, total RNA of Haloxylon ammodendron is extracted and reversely transcribed into cDNA, and a nucleotide sequence as shown in SEQ ID NO: 1 is amplified by taking Haloxylon ammodendron cDNA as a template HaCLA1 fragment; S12, double enzyme digestion to prepare pTRV2 linearized vector; S13, recover the target fragment by DNA gel recovery kit HaCLA1 and pTRV2 linearized vector; S14, recombining the target fragment HaCLA1 and the pTRV2 linearized vector to obtain a recombination product, and connecting the recombination reaction solution; S15, transforming the recombination product reaction solution into DH5a E. coli competent cells to obtain a recombination vector pTRV2- HaCLA1 ; S2, recombination of the vector to transform Agrobacterium: extract pTRV1, pTRV2- HaCLA1 plasmid and transfer it into the melted GV3101 Agrobacterium competent, select positive clone bacteria, get pTRV1 and pTRV2- HaCLA1 GV3101 Agrobacterium strain; S3, infection of P. polyphylla var. humilis; S31, preparation of transfection bacterial liquid; S32, infection of P. polyphylla var. humilis by tobacco amplification virus homogenate.

2. The method of silencing a Gossypium arboreum gene by virus induction according to claim 1, wherein, The system of the double enzyme digestion in S12 is as follows: 5 μL of 10×FastDigest buffer, 1 μL of EcoR I and 1 μL of Kpn I, 1 μL of pTRV2 empty vector, and sterile water is added to make up the volume to 20 μL, and the enzyme digestion condition is 37°C for 30 min.

3. The method of silencing a P. somnifera gene by virus induction as claimed in claim 1, wherein, The reaction system for recombination described in S14 is 10 μL: 5 μL of 2x Uniclone Seamless Cloning Mix, 2 μL of linearized vector pTRV2, HaCLA1 3 μL, under temperature conditions of 50°C for 30 min.

4. The method of silencing a Gossypium arboreum gene by virus induction according to claim 1, wherein, The method of recombination product in S15 is transformed by E. coli competent cell DH5α as follows: E. coli competent cell DH5α is taken, 10 μL of the ligation recombination reaction liquid prepared in S15 is added, and the mixture is mixed uniformly and placed on ice for 30 min; after the placement is finished, the mixture is placed in a 42°C metal bath for reaction for 45 s, and then placed on ice for 2 min; then, 700 μL of sterilized anti-LB liquid medium is added, and the mixture is placed in a 37°C incubator, and recovered for 1 h at a rotation speed of 220 rpm; then, 100 μL is taken and coated on LB solid medium with a kanamycin sulfate resistance of 50 μg / ml, and cultured in a 37°C incubator overnight.

5. The method of silencing a Gossypium arboreum gene by virus induction according to claim 1, wherein, The preparation of transfection bacterial liquid in S31 comprises the following steps: S311, Activation: Activate the pTRV1 and pTRV2 prepared in S2. HaCLA1 Agrobacterium strain GV3101 was streaked on YEB solid medium to obtain activated Agrobacterium; S312, small shaking of bacteria: the activated Agrobacterium monoclonal is taken in 1 mL of YEB liquid medium, and cultured overnight at 28°C on a shaking table at a rotation speed of 180 rpm; S313, Agitate the Agrobacterium culture at 28°C, 180 rpm until OD 600 = 0.7-1.

2. S314, processing the bacterial strain: centrifuge the product prepared in S313 at 5000 rpm for 10 min, discard the supernatant and enrich the bacterial colonies, add resuspension liquid to resuspend the enriched bacterial colonies to OD 600 = 0.6-0.8, stand still at room temperature for 2-3 h, and then mix pTRV1 and pTRV2- HaCLA1 Mix by equal volume in a ratio of 1:1 to obtain the bacterial liquid for transfection.

6. The method of silencing a P. somnifera gene by virus induction as claimed in claim 5, wherein, The YEB solid medium in S311 and the YEB liquid medium in S312 and S313 all add 50 μg / mL of rifampicin and 50 μg / mL of kanamycin sulfate.

7. The method of silencing a P. somnifera gene by virus induction as claimed in claim 5, wherein, The resuspension liquid in S314 is obtained by uniformly mixing MS liquid medium, 10 mmol / L MgCL2, 10 mmol / L MES and 150 μmol / L AS.

8. The method of silencing a gene in Gossypium stocksii virus-induced according to claim 1, wherein, The method of infection of P. polyphylla var. humilis by tobacco amplification virus homogenate in S32 is as follows: the transfection bacterial liquid prepared in S31 is slowly injected through a wound on a tobacco leaf, and after 7 d of culture, 1 g of the tobacco leaf is weighed, the leaf is cut into pieces and placed in a sterilized mortar, 1 mL of 0.2 mmol / L sodium phosphate buffer with a pH value of 7.2 is added, the tobacco leaf is ground into a liquid homogenate in the mortar, and after filtration, virus liquid is obtained; the virus liquid is diluted with sterile water at a ratio of 1:40 to obtain virus infection liquid; four-week-old P. polyphylla var. humilis plants are soaked in the virus infection liquid in a dark environment for 48 h, the temperature is maintained at 22-24°C, and then the plants are transferred to a culture box to observe the phenotype of the P. polyphylla var. humilis plants.

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