Virus-induced haloxylon ammodendron gene silencing method

Abstract

The invention relates to the technical field of gene engineering, in particular to a virus-induced haloxylon ammodendron gene silencing method. The invention provides a method for silencing a haloxylon ammodendron gene through virus induction. The method comprises the following steps: constructing a VIGS vector, transforming agrobacterium tumefaciens, preparing a transfection bacterial solution, infecting a haloxylon ammodendron plant through virus homogenate and the like. A haloxylon ammodendron gene silencing system is established by using the virus vector modified by the tobacco embrittlement virus, the gene silencing mediated by virus homogenate is faster and more efficient, and the expression quantity of the haloxylon ammodendron HaCLA1 gene after silencing is obviously reduced. According to the invention, a VIGS gene silencing technology is actually applied to haloxylon ammodendron for the first time, a set of efficient haloxylon ammodendron gene silencing system is established, target genes of plants can be efficiently silenced in a short time, the operation is simple and convenient, the efficiency is high, the experimental period is short, and the cost is low. Meanwhile, the HaCLA1 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] The invention relates to the technical field of genetic engineering, and in particular to a method for silencing Haloxylon ammodendron gene induced by virus. Background Art

[0002] Haloxylon ammodendron (CA Mey.) Bunge is a super-xerophytic shrub or small tree of the genus Haloxylon in the family Amaranthaceae, 1 to 9 meters high, also known as saltwood, saosuo tree, Haloxylon firewood, etc. Haloxylon ammodendron is one of the most widely distributed desert plants in desert areas. It is resistant to high temperature and drought, salt and alkali, barrenness, and drought. It has a strong adaptability to the harsh desert environment. It is an ideal material for studying plant stress resistance mechanisms. It is also the largest tree species for sand fixation and afforestation in arid areas and is known as the "Desert Guardian". Haloxylon ammodendron has hard wood and is a good firewood. The assimilating branches of the current year have a high crude protein content and are rich in nutrients. They are excellent feed for livestock such as camels and sheep. The precious Chinese medicinal material Cistanche deserticola, known as the "desert ginseng", parasitizes on its roots. Therefore, Haloxylon ammodendron has important ecological value, feeding value and economic value.

[0003] Among the existing genomic research methods, virus-induced gene silencing (VIGS) is a relatively mature transgenic technology, which is widely used in monocotyledonous and dicotyledonous plants, such as tomatoes, tobacco, cotton, peppers, Arabidopsis, peppers, potatoes, corn, wheat, petunias, etc. VIGS can achieve targeted gene silencing in contemporary plants through viral vector inoculation. VIGS was first used to explain the defense mechanism of plants caused by virus infection, and this phenomenon is common in plants. VIGS utilizes the inherent RNA interference and viral immune response mechanisms of plants.

[0004] There are currently very few reports in the literature on the establishment of a stable regeneration system and genetic transformation system for Haloxylon ammodendron. It is difficult to study the functions of Haloxylon ammodendron genes using T-DNA insertion mutagenesis, RNA interference (RNAi), and CRISPR / Cas9 systems. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for silencing Haloxylon ammodendron genes induced by virus in view of the deficiencies of the above-mentioned prior art. A Haloxylon ammodendron silencing system is established by using tobacco rattle virus (TRV) as a viral vector through the steps of constructing a VIGS vector, transforming with Agrobacterium, preparing bacterial solution and virus homogenate for transfection, infecting Haloxylon ammodendron plants, etc. The gene silencing mediated by virus homogenate is faster and more efficient. After silencing, the expression level of Haloxylon ammodendron HaCLA1 gene is significantly reduced. The method can efficiently silence the target gene of the plant in a short time, and the operation is simple, efficient, short in experimental cycle and low in cost.

[0006] The present invention provides a method for silencing Haloxylon ammodendron gene by virus induction, comprising the following steps: S1. VIGS vector construction: S11, extracting total RNA from Haloxylon ammodendron and reversely transcribing it into cDNA, and using the Haloxylon ammodendron cDNA as a template to amplify a HaCLA1 fragment containing a nucleotide sequence as shown in SEQ ID NO: 1; S12, double enzyme digestion to prepare pTRV2 linearized vector; S13, recovering the target fragment HaCLA1 and the pTRV2 linearized vector using a DNA gel recovery kit; S14, recombining the target fragment HaCLA1 and the pTRV2 linearized vector, and connecting the obtained recombinant product to the recombinant reaction solution; S15, the recombinant product reaction solution was transformed into DH5α Escherichia coli competent cells to obtain the recombinant vector pTRV2-HaCLA1; S2. Transformation of recombinant vector into Agrobacterium: extract pTRV1, pTRV2, and pTRV2-HaCLA1 plasmids and transfer them into melted GV3101 Agrobacterium competent cells, respectively, screen positive clones, and obtain GV3101 Agrobacterium strains of pTRV1, pTRV2, and pTRV2-HaCLA1; S3, infection of Haloxylon ammodendron: S31, preparing bacterial solution for transfection; S32. Use tobacco amplified virus homogenate to infect Haloxylon ammodendron.

[0007] According to a virus-induced method for silencing the H. ammodendron gene provided by the present invention, the double enzyme digestion system described in S12 is: 5 μL of 10×FastDigest buffer, 1 μL each of EcoRⅠ and Kpn I, 1 μL of pTRV2 empty vector, the volume is supplemented to 20 μL with sterile water, and the enzyme digestion conditions are 37°C for 30 min.

[0008] According to a virus-induced method for silencing the H. ammodendron gene provided by the present invention, the recombinant reaction system described in S14 is 10 μL in total: 5 μL of 2×Uniclone Seamless Cloning Mix, 2 μL of linearized vector pTRV2, and 3 μL of HaCLA1, and the reaction is carried out at a temperature of 50°C for 30 min.

[0009] According to a method for virus-induced silencing of H. ammodendron genes provided by the present invention, the method for transforming the recombinant product in S15 through Escherichia coli competent cells DH5α is as follows: take Escherichia coli competent cells DH5α, add 10 μL of the ligation and recombination reaction solution prepared in S15, gently mix, and place it on ice for 30 min. After the standing period, place it in a 42°C metal bath for reaction for 45 s and then immediately place it on ice for 2 min, then add 700 μL of sterilized antibiotic-free LB liquid culture medium, then place it in a 37°C incubator, resuscitate it at 220 rpm for 1 h, then aspirate 100 μL and apply it to LB solid culture medium with kanamycin sulfate 50 μg / mL resistance, and culture it in an incubator at 37°C overnight.

[0010] According to a virus-induced method for silencing Haloxylon ammodendron gene provided by the present invention, the preparation of bacterial solution for transfection in S31 comprises the following steps: S311, activation: streak the GV3101 Agrobacterium strains of pTRV1, pTRV2 and pTRV2-HaCLA1 prepared in S2 on YEB solid medium to obtain activated Agrobacterium; S312, small shaker strain: pick a single clone of Agrobacterium after activation and place it in 1 mL YEB liquid medium, and culture it overnight at 28°C in a shaker at 180 rpm; S313, shake the bacteria: transfer 100 μL of the Agrobacterium cultured in S312 to 10 mL of YEB liquid medium, and culture at 28°C, 180 rpm until OD600 = 0.7-1.2; S314. Treat bacterial strains: Centrifuge the product prepared in S313 at 5000 rpm for 10 min, discard the supernatant to enrich the colonies, add resuspension solution to resuspend the enriched colonies to OD600 = 0.6-0.8, and let it stand at room temperature for 2-3 h. Mix pTRV1 with pTRV2 and pTRV2-HaCLA1 in an equal volume at a ratio of 1:1 to obtain a bacterial solution for transfection.

[0011] According to a virus-induced method for silencing the H. ammodendron gene 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 in S311 and the YEB liquid culture medium in S312 and S313.

[0012] According to a virus-induced method for silencing the H. 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.

[0013] According to a method for virus-induced silencing of Haloxylon ammodendron genes provided by the present invention, the method of using tobacco amplified virus homogenate to infect Haloxylon ammodendron in S32 is: slowly inject the transfection bacterial solution prepared in S31 through the wound on the tobacco leaves, and after culturing for 7 days, weigh 1 g of tobacco leaves, cut the leaves into pieces and put them into a sterilized mortar, then add 1 mL of sodium phosphate buffer with a pH value of 7.2 and a concentration of 0.2 mmol / L, grind the tobacco leaves into a liquid homogenate in the mortar, and obtain the virus liquid after filtration, dilute the virus liquid with sterile water at a ratio of 1:40 to obtain a virus infection liquid, soak four-week-old Haloxylon ammodendron plants in the virus infection liquid and culture them in a dark environment for 48 h, the temperature is maintained at 22-24°C, and then transfer them to an incubator to observe the phenotype of the Haloxylon ammodendron plants. Compared with the prior art, the present invention has the following advantages: The present invention provides a method for silencing Haloxylon ammodendron genes induced by virus, the method comprising the steps of constructing a VIGS vector, transforming with Agrobacterium, preparing a bacterial solution for transfection and a virus homogenate to infect Haloxylon ammodendron plants, etc. In the present invention, a Haloxylon ammodendron silencing system is established by using tobacco rattle virus as a virus vector, and the results show that both the transfection bacterial solution and the virus homogenate can be used to infect Haloxylon ammodendron plants to obtain a gene silencing phenotype, but the virus homogenate-mediated gene silencing is faster and more efficient, and qPCR is used to detect the expression of Haloxylon ammodendron HaCLA1 gene at the same time. Compared with the control group, the expression of Haloxylon ammodendron HaCLA1 gene is significantly reduced after silencing. The present invention is the first to apply the VIGS gene silencing technology to Haloxylon ammodendron, and establish a set of efficient Haloxylon ammodendron gene silencing system, which can efficiently silence the target gene of the plant in a short time, is simple and efficient to operate, has a short experimental cycle and is low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 is the pTRV2-HaCLA1 plasmid map; Figure 2 is the agarose gel electrophoresis image of PCR for cloning HaCLA1 gene; Figure 3 is the flow chart of TRV virus-mediated VIGS in H. ammodendron; Figure 4 This is the phenotype of the Haloxylon ammodendron control plant and the plant after the HaCLA1 gene was silenced; Figure 5This is the assimilation branch phenotype of the Haloxylon ammodendron control plant and the plant after the HaCLA1 gene was silenced; Figure 6 qPCR was used to detect the changes in the expression levels of the Haloxylon ammodendron HaCLA1 gene in the control strain and the silenced strain. DETAILED DESCRIPTION Example

[0016] The planting method of the raw material Haloxylon ammodendron used in this embodiment is as follows: the seeds of Haloxylon ammodendron were collected in Turpan, Xinjiang, and the Haloxylon ammodendron seeds with full grains and similar shapes and sizes were selected, soaked and disinfected with 75% alcohol for 60 seconds, vortexed and shaken to mix thoroughly, filtered, rinsed with sterile water, shaken and filtered 3 times, soaked and shaken with sodium hypochlorite stock solution, filtered after standing for 13 minutes, rinsed with sterile water, shaken and filtered 6 times, and then the clean Haloxylon ammodendron seeds were inoculated in a sterile 250 ML tissue culture bottle MS solid culture medium, the distance between the seeds was kept consistent, and the culture temperature was 28°C, the relative air humidity was 60-65%, the light conditions were 8h / 16h and the light intensity was 5000LX. The light was placed in a light incubator for culture to obtain Haloxylon ammodendron assimilated branches.

[0017] Extraction and detection of total RNA from Haloxylon ammodendron: Haloxylon ammodendron assimilating branches were used as raw materials, which were quickly frozen in liquid nitrogen and then fully ground. RNA was extracted using an RNA extraction kit (Shanghai Sangon Biotechnology Co., Ltd., B518661) and reverse transcribed into cDNA using a reverse transcription kit (Applied Biological Materials Inc (ABM), G592).

[0018] This embodiment provides a method for silencing the Haloxylon ammodendron gene induced by virus, and the specific steps are as follows: S1. VIGS vector construction S11. Based on the gene sequence of Hacla1 (cloroplastos alterados 1 gene), the upstream and downstream of the pTRV2 vector sequence and the restriction sites of EcoRI and KpnI were designed to construct the recombinant vector pTRV2-HaCLA1. In order to reduce the introduction of amplification mutations, high-fidelity polymerase 2×Phanta Max Master Mix (Dye Plus) (Vazyme #P525) was used for amplification; The nucleotide sequence of HaCLA1 is shown in SEQ ID NO: 1, and the primers thereof 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; Preparation of S12 and pTRV2 linearized vectors The linearized vector was prepared by enzyme digestion. The double enzyme digestion method was used to completely linearize the vector and reduce the transformation background. The extracted plasmid pTRV2 was double-digested with Thermo Scientific FastDigest EcoR I and Kpn I respectively; EcoR I and Kpn I double enzyme digestion system: 5 μL of 10× FastDigest buffer, 1 μL of EcoRⅠ and Kpn I, 1 μL of pTRV2 empty vector, add sterile water to make up the volume to 20 μL, and the enzyme digestion condition is 37℃ for 30 min; S13, gel recovery of target fragments and vectors The target fragment HaCLA1 and the pTRV2 vector were subjected to electrophoresis, and the gel containing the target fragment HaCLA1 and the linearized vector pTRV2 were cut out respectively. The target fragment and the linearized vector were purified according to the method of the DNA gel recovery kit (Meiji); Figure 2 This is the agarose gel electrophoresis diagram of PCR for cloning the HaCLA1 gene, where M is 2K DNA Marker and 1 is HaCLA1; S14. Recombination reaction According to the Uniclone One Step Seamless Cloning Kit (Jinsha), the recombination reaction system of 10 μL was as follows: 2× Uniclone Seamless Cloning Mix 5 μL, linearized vector pTRV2 2 μL, HaCLA1 3 μL, and the reaction was carried out at 50°C for 30 min. S15. Transformation of recombinant products Take out the competent E. coli DH5α cells from the -80℃ refrigerator and place them on ice. When the E. coli melts, add 10 μL of the ligation and recombination reaction solution, gently stir, and place it on ice for 30 min. After the standing, the centrifuge tube was placed in a 42°C metal bath, and immediately placed on ice after reacting for 45 seconds for 2 minutes. The centrifuge tube was then transferred to a clean bench, and 700 μL of sterilized, non-antibiotic LB liquid medium was added to the centrifuge tube. The centrifuge tube was placed in a 37°C incubator and revived at 220 rpm for 1 hour. 100 μL was then applied to a LB solid medium containing 50 μg / mL kanamycin sulfate resistance and cultured overnight in a 37°C incubator. S16. Gene fragment verification Monoclonal colonies were picked from the LB solid medium containing kanamycin resistance cultured overnight, and the bacterial liquid PCR was performed to verify the colonies. After the band size was correct, they were sequenced at Shanghai Sangon Biotechnology Co., Ltd. to obtain the recombinant vector pTRV2-HaCLA1. S2. Transformation of recombinant vector into Agrobacterium Extract pTRV1, pTRV2, and pTRV2-HaCLA1 plasmids and transfer them into the melted GV3101 Agrobacterium competent cells, screen positive clones, and pick Agrobacterium monoclonal clones grown on the culture medium according to the instructions of the GV3101 Agrobacterium competent cells of the Biotechnology Industry. Identify positive monoclonal clones by PCR of the bacterial solution, and store the bacteria in a -80℃ refrigerator for later use; S3, infection of Haloxylon ammodendron S31. Prepare bacterial solution for transfection to infect Haloxylon ammodendron ① Activation: streak the GV3101 Agrobacterium strains of pTRV1, pTRV2, and pTRV2-HaCLA1 obtained earlier on YEB solid medium; YEB solid medium contains 50 μg / mL rifampicin and 50 μg / mL kanamycin sulfate. The preparation method of YEB solid medium is as follows: 31.24 g YEB medium is measured, and the volume is made up to 1000 mL with deionized water, and the medium is stirred evenly, and sterilized at 121℃ for 20 min. The medium is cooled to about 55℃, and then 1 mL of 50 mg / mL rifampicin and 0.5 mL of 100 mg / mL kanamycin are added, and the culture dish is poured and stored in a refrigerator at 4℃. ② Small shaking bacteria: Pick a single clone of Agrobacterium tumefaciens and place it in 1 mL YEB liquid medium (rifampicin 50 μg / mL + kanamycin sulfate 50 μg / mL), and culture it overnight at 28°C in a shaking incubator at 180 rpm; ③ Shake the culture: transfer 100 μL of cultured Agrobacterium to 10 mL YEB liquid medium (rifampicin 50 μg / mL + kanamycin sulfate 50 μg / mL), and culture at 28°C, 180 rpm until OD600 = 0.7-1.2; ④ Treat bacteria: collect bacteria, centrifuge at 5000 rpm for 10 min, discard the supernatant, enrich colonies, add the prepared resuspension solution to resuspend the enriched colonies to OD600 = 0.6-0.8, let 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; Resuspension (100 mL): Take 100 μL of 150 mM acetosyringone solution for sterilization, 2 mL of 10 mM MgCL2 solution, 2 mL of 10 mM MES solution, and add 1×MS liquid culture medium to make up to 100 mL; ⑤ Soak the four-week-old Haloxylon ammodendron plants in the transfection solution, culture them in a dark environment for 48 hours, maintain the temperature at 22-24°C, and then transfer them to an incubator to observe the phenotype of the Haloxylon ammodendron plants; S32. Infection of Haloxylon ammodendron with Tobacco-amplified Virus Homogenate Use a syringe to slowly inject the transfection bacterial solution in step S31 through the wound on the tobacco leaf, and continue to culture for 7 days, weigh 1 g of tobacco leaves containing virus particles injected with Agrobacterium resuspension and cultured for 7 days, cut the leaves into pieces and put them into a sterilized mortar, then add 1 mL of 0.2 M sodium phosphate buffer (PB) with a pH of 7.2, grind the tobacco leaves into a liquid homogenate in the mortar, filter the homogenate into a beaker with four layers of gauze to remove large substances, divide the filtered liquid into 2 mL centrifuge tubes, and store at -20°C for later use; dilute the prepared virus solution with sterile water at a ratio of 1:40 to form a virus infection solution, soak the four-week-old Haloxylon ammodendron plants in the virus infection solution and culture them in a dark environment for 48 hours, and maintain the temperature at 22-24°C, and then transfer them to an incubator to observe the phenotype of the Haloxylon ammodendron plants.

[0019] Gene expression detection: Continue to observe the albino phenomenon of the assimilating branches of Haloxylon ammodendron injected with pTRV2-HaCLA1. The albino phenomenon indicates that the expression of the HaCLA1 gene in Haloxylon ammodendron has been successfully reduced, and Haloxylon ammodendron cannot synthesize chlorophyll normally, indicating that the silencing system has been successfully constructed. Once the Haloxylon ammodendron injected with pTRV2-HaCLA1 showed albinism, the Haloxylon ammodendron assimilated branches with pTRV2-HaCLA1 and pTRV2 were picked, quickly frozen in liquid nitrogen and ground, and RNA was extracted using an RNA extraction kit (Shanghai Biotechnology Co., Ltd., B518661). The RNA was reverse transcribed into cDNA using a reverse transcription kit (Applied Biological Materials Inc (ABM), G592), and the expression of the HaCLA1 gene was quantitatively detected by real-time fluorescence.

[0020] The fluorescence quantitative PCR reaction system is as follows: in each 20 uL reaction system, there are 10 μL of BlasTaq™ 2× qPCRMasterMix, 0.5 μL of upstream and downstream primers, 2 μL of template, and the volume is supplemented to 20 μL with sterile water. The internal reference gene is 18SrRNA; the nucleotide sequence of primer 18SrRNA-F is shown in SEQ ID NO: 4, and the nucleotide sequence of primer 18SrRNA-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; The fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing and extension at 60°C for 60 s, and 40 cycles.

[0021] Real-time fluorescence quantitative PCR data analysis method The relative expression of the target gene in this experiment was analyzed using the 2-△△CT method. The relative expression of the target gene was calculated using the formula: △CT target gene = CT target gene - CT reference gene △CT control target gene = CT control target gene - CT control internal reference gene △△CT=△CT target gene-△CT control target gene Relative expression of target gene = 2-△△CT = 2-(△CT target gene-△CT control target gene) Figure 4 The phenotypes of the Haloxylon ammodendron control plant and the plant after the HaCLA1 gene was silenced, where the left side is the control sample and the right side is the albino plant after the HaCLA1 gene was silenced; Figure 5 This is the phenotype of assimilated branches of the Haloxylon ammodendron control plant and the plant after the HaCLA1 gene was silenced, where the left side is the assimilated branch of the control plant, and the right side is the assimilated branch of the albino plant after the HaCLA1 gene was silenced; Figure 6 qPCR was used to detect the changes in the expression levels of the Haloxylon ammodendron HaCLA1 gene in the control strain and the silenced strain. It can be seen that the expression level of the Haloxylon ammodendron HaCLA1 gene was significantly reduced after silencing.

[0022] In this example, tobacco rattle virus was used as a viral vector to establish a Haloxylon ammodendron silencing system. The results showed that the gene silencing phenotype could be obtained by infecting Haloxylon ammodendron plants with transfection bacterial solution and virus homogenate, but the gene silencing mediated by virus homogenate was faster and more efficient. At the same time, qPCR was used to detect the expression of Haloxylon ammodendron HaCLA1 gene. Compared with the control group, the expression of Haloxylon ammodendron HaCLA1 gene was significantly reduced after silencing. This example is the first to apply VIGS gene silencing technology in Haloxylon ammodendron, and establish a set of efficient Haloxylon ammodendron gene silencing system, which can effectively silence the target gene of the plant in a short time, is simple and efficient to operate, has a short experimental cycle and is low in cost.

[0023] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for silencing Haloxylon ammodendron gene by virus induction, characterized in that: The following steps are involved: S1. VIGS vector construction: S11, extracting total RNA of Haloxylon ammodendron and reversely transcribing it into cDNA, using Haloxylon ammodendron cDNA as a template to amplify a gene containing the nucleotide sequence shown in SEQ ID NO: 1 HaCLA1 Fragment; S12, double enzyme digestion to prepare pTRV2 linearized vector; S13. Recover the target fragment using a DNA gel recovery kit HaCLA1 and pTRV2 linearized vector; S14, recombinant target fragment HaCLA1 and pTRV2 linearized vector, and the obtained recombinant product was connected to the recombinant reaction solution; S15, the recombinant product reaction solution was transformed into DH5α Escherichia coli competent cells to obtain the recombinant vector pTRV2- HaCLA1 ; S2. Transformation of Agrobacterium with recombinant vector: Extraction of pTRV1, pTRV2, and pTRV2- HaCLA1 The plasmids were transferred into the melted GV3101 Agrobacterium competent cells, and positive clones were screened to obtain pTRV1, pTRV2 and pTRV2- HaCLA1 GV3101 Agrobacterium strain; S3, infection of Haloxylon ammodendron: S31, preparing bacterial solution for transfection; S32. Use tobacco amplified virus homogenate to infect Haloxylon ammodendron.

2. The method for silencing the Haloxylon ammodendron gene induced by virus according to claim 1, characterized in that: The double enzyme digestion system described in S12 is: 5 μL of 10×FastDigest buffer, 1 μL each of EcoRⅠ and Kpn I, 1 μL of pTRV2 empty vector, and the volume is supplemented to 20 μL with sterile water. The enzyme digestion conditions are 37°C for 30 min.

3. The method for silencing the Haloxylon ammodendron gene induced by virus according to claim 1, characterized in that: The reaction system for the recombination described in S14 is 10 μL in total: 2× Uniclone Seamless Cloning Mix 5 μL, linearized vector pTRV2 2 μL, HaCLA1 3 μL, react at 50 °C for 30 min.

4. The method for silencing Haloxylon ammodendron gene by virus induction according to claim 1, characterized in that: The method for transforming the recombinant product described in S15 by using Escherichia coli competent cells DH5α is as follows: take Escherichia coli competent cells DH5α, add 10 μL of the ligation and recombination reaction solution prepared in S15, gently mix, and place it on ice for 30 min. After the standing period, place it in a 42°C metal bath for reaction for 45 s and then immediately place it on ice for 2 min, then add 700 μL of sterilized antibiotic-free LB liquid culture medium, then place it in a 37°C incubator, resuscitate it at 220 rpm for 1 h, then aspirate 100 μL and apply it to LB solid culture medium with kanamycin sulfate 50 μg / ml resistance, and culture it in an incubator at 37°C overnight.

5. The method for silencing the Haloxylon ammodendron gene induced by virus according to claim 1, characterized in that: The preparation of the bacterial solution for transfection in S31 comprises the following steps: S311, activation: pTRV1, pTRV2 and pTRV2- HaCLA1 The GV3101 Agrobacterium strain was streaked on YEB solid medium to obtain activated Agrobacterium; S312, small shaker strain: pick a single clone of Agrobacterium after activation and place it in 1 mL YEB liquid medium, and culture it overnight at 28°C in a shaker at 180 rpm; S313, large shaking culture: transfer 100 μL of Agrobacterium cultured in S312 to 10 mL of YEB liquid medium and culture at 28°C and 180 rpm until OD 600 =0.7~1.2; S314. Treat the bacteria: Centrifuge the product prepared in S313 at 5000 rpm for 10 min, discard the supernatant to enrich the colonies, and add the resuspension solution to resuspend the enriched colonies until the OD 600 = 0.6~0.8, and let stand at room temperature for 2~3 h. Then, pTRV1 and pTRV2, pTRV2- HaCLA1 Mix equal volumes in a 1:1 ratio to obtain bacterial solution for transfection.

6. The method for silencing the Haloxylon ammodendron gene induced by virus according to claim 5, characterized in that: 50 μg / mL of rifampicin and 50 μg / mL of kanamycin sulfate were added to the YEB solid culture medium described in S311 and the YEB liquid culture medium described in S312 and S313.

7. The method for silencing the Haloxylon ammodendron gene induced by virus according to claim 5, characterized in that: 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.

8. The method for silencing the Haloxylon ammodendron gene induced by virus according to claim 1, characterized in that: The method of using tobacco amplified virus homogenate to infect Haloxylon ammodendron as described in S32 is: slowly inject the transfection bacterial solution prepared in S31 through the wound on the tobacco leaves, and after culturing for 7 days, weigh 1 g of tobacco leaves, cut the leaves into pieces and put them into a sterilized mortar, then add 1 mL of sodium phosphate buffer with a pH value of 7.2 and a concentration of 0.2 mmol / L, grind the tobacco leaves into a liquid homogenate in the mortar, and obtain the virus solution after filtration, dilute the virus solution with sterile water at a ratio of 1:40 to become a virus infection solution, soak four-week-old Haloxylon ammodendron plants in the virus infection solution and culture them in a dark environment for 48 h, maintaining the temperature at 22-24°C, and then transfer them to an incubator to observe the phenotype of the Haloxylon ammodendron plants.

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