Application of dsRNA targeting both gawky and CHS genes of Bemisia tabaci in biological control of Bemisia tabaci
Patent Information
- Application Number
- CN202611081456.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-21
- Publication Date
- 2026-10-09
AI Technical Summary
[0005]然而，单一靶基因的RNAi效率有时不够稳定，且存在害虫产生耐受性的风险
（1）本发明基于RNA干扰技术，首次将烟粉虱Gawky基因与CHS基因作为双靶点，构建了融合dsRNA的表达体系。实验结果表明，该双靶点融合dsRNA可同时干扰烟粉虱体内两条关键功能通路，相较于单靶点（dsGawky或dsCHS）处理，对烟粉虱成虫、若虫及卵均表现出显著的协同增效致死效应，有效提升了RNAi介导的害虫防治效率，降低了害虫对单一靶标产生耐受性的风险，在烟粉虱的生物防治领域具有重要的应用前景。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural pest control technology, specifically involving the application of dsRNA fusion of the Gawky gene and CHS gene of whitefly in the biological control of whitefly. Background Technology
[0002] The whitefly (Bemisia tabaci), belonging to the family Amyliidae in the order Hemiptera, is a globally invasive pest with populations distributed throughout tropical, subtropical, and adjacent temperate regions worldwide. Whiteflies have a wide host range, damaging various crops including cotton, vegetables, and flowers. Their methods of damage include directly sucking plant sap, secreting honeydew which induces sooty mold, and acting as a vector for plant viruses, causing billions of dollars in economic losses to global agricultural production annually. In the past two decades, invasive species such as the B-type whitefly (now known as MEAM1 cryptospora) and the Q-type whitefly (now known as MED cryptospora) have spread widely worldwide, replacing native whiteflies in many invasive areas and making them a major agricultural pest of close global concern.
[0003] Currently, chemical control remains the primary method for controlling whiteflies. However, due to the long-term and extensive use of chemical agents, whiteflies have developed varying degrees of resistance to many commonly used insecticides. Monitoring data shows that whitefly populations in the field have exhibited moderate to high levels of resistance to agents such as spirotetramat and cyantraniliprole. Over-reliance on chemical pesticides not only leads to increased control costs and decreased efficacy but also causes environmental pollution and food safety risks. Therefore, there is an urgent need to develop new, green, efficient, and environmentally friendly whitefly control technologies.
[0004] RNA interference (RNAi) is a phenomenon of homologous mRNA-specific degradation induced by double-stranded RNA (dsRNA). In recent years, it has been widely used in the functional study of target genes for pest control and the development of novel insecticides. This technology interferes with the expression of key genes in pests, leading to inhibited growth and development or death of pests, and has advantages such as strong targeting and good environmental compatibility.
[0005] However, the efficiency of RNAi targeting a single gene is sometimes unstable and carries the risk of pests developing tolerance. A dual-gene target strategy, by simultaneously interfering with key genes in two different functional pathways, holds promise for producing a synergistic lethal effect, thereby improving the efficiency and reliability of RNAi control. Based on this, this invention provides a dual-target fusion dsRNA targeting the Gawky and CHS genes of the whitefly and its application in the biological control of whiteflies. Summary of the Invention
[0006] To address the problems existing in the prior art, the purpose of this invention is to provide the application of dsRNA fusion targeting two genes, Gawky and CHS, in the biological control of whiteflies.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: Application of dsRNA targeting the Gawky and CHS genes of the whitefly in the biological control of the whitefly, wherein the nucleic acid sequence of the Gawky gene is shown in SEQ ID NO.1 and the nucleic acid sequence of the CHS gene is shown in SEQ ID NO.2.
[0008] Based on the above scheme, RNA interference was used to interfere with the Gawky and CHS genes of the whitefly, thereby increasing the mortality rate of the whitefly.
[0009] Based on the above scheme, the whitefly mentioned is either type Q or type B.
[0010] A method for controlling whiteflies involves interfering with the Gawky and CHS genes of whiteflies via RNA interference, thereby increasing the mortality rate of whiteflies. The nucleic acid sequence of the Gawky gene is shown in SEQ ID NO.1, and the nucleic acid sequence of the CHS gene is shown in SEQ ID NO.2.
[0011] Based on the above solution, the specific steps of the method for controlling whiteflies are as follows: (1) Prepare a recombinant vector expressing dual-target dsRNA of the whitefly Gawky gene and CHS gene, and transform it into the expression strain to induce expression; (2) The bacterial cells that induce the expression of dsRNA were prepared into a bacterial solution and sprayed on plants, so that whiteflies would feed on the sprayed plants, thereby increasing the mortality rate of whiteflies.
[0012] Based on the above scheme, the recombinant vector expressing the dual-target fusion dsRNA of the whitefly Gawky gene and CHS gene was prepared by the following method: Primers for preparing dsRNA of Gawky and CHS genes were used, and cDNA reverse transcribed from total RNA of whiteflies was used as a template for PCR amplification. The amplified fragment was ligated into an expression vector to obtain a recombinant vector expressing dsRNA of dual gene target sites of whiteflies Gawky and CHS genes. The primers for preparing Gawky gene dsRNA are shown in SEQ ID NO.4 and SEQ ID NO.5; The primers for preparing CHS gene dsRNA are shown in SEQ ID NO.6 and SEQ ID NO.7.
[0013] Based on the above scheme, the whitefly mentioned is either type Q or type B.
[0014] An agent for controlling whiteflies contains recombinant bacteria expressing a fusion dsRNA of the whitefly Gawky gene and the CHS gene, wherein the nucleic acid sequence of the Gawky gene is shown in SEQ ID NO.1 and the nucleic acid sequence of the CHS gene is shown in SEQ ID NO.2.
[0015] Based on the above scheme, the preparation method of the agent for controlling whiteflies is as follows: (1) Using primers for preparing Gawky and CHS gene dsRNA, and cDNA reverse transcribed from total RNA of whitefly as template, PCR amplification was performed; the amplified fragment was ligated into the expression vector to obtain a recombinant vector expressing dual target dsRNA of Gawky and CHS genes of whitefly. (2) Transform the recombinant vector expressing dual-target dsRNA of the whitefly Gawky gene and CHS gene into the expression strain to induce expression; (3) Centrifuge the bacterial culture after induction to separate the bacterial cells, discard the supernatant, resuspend the bacterial cells in PBS solution, mix well, centrifuge, discard the supernatant, repeat the PBS washing step twice to obtain the preparation for the control of whiteflies.
[0016] Based on the above scheme, the primers for preparing Gawky gene dsRNA are shown in SEQ ID NO.4 and SEQ ID NO.5; the primers for preparing CHS gene dsRNA are shown in SEQ ID NO.6 and SEQ ID NO.7.
[0017] Advantages of the technical solution of this invention (1) Based on RNA interference technology, this invention is the first to construct an expression system fused with dsRNA, using the Gawky and CHS genes of the whitefly as dual targets. Experimental results show that the dual-target fused dsRNA can simultaneously interfere with two key functional pathways in the whitefly. Compared with single-target (dsGawky or dsCHS) treatment, it shows a significant synergistic lethal effect on adult whiteflies, nymphs, and eggs, effectively improving the efficiency of RNAi-mediated pest control and reducing the risk of pests developing tolerance to a single target. It has important application prospects in the field of biological control of whiteflies.
[0018] (2) This invention uses the L4440 plasmid as a vector and Escherichia coli HT115(DE3) strain as a host to achieve large-scale, low-cost synthesis of Gawky-CHS dual-target fusion dsRNA through IPTG-induced expression. Compared with commercially available dsRNA in vitro transcription kits, the engineered bacteria fermentation production process of this invention significantly reduces the synthesis cost of dsRNA and is easy to operate, providing a feasible technical solution for the large-scale production and field application of RNAi preparations.
[0019] (3) This invention uses direct spraying of bacterial solution onto plant leaves, which is simple to operate and does not require complicated dsRNA purification steps, making it suitable for large-scale field application. At the same time, this control method does not rely on chemical pesticides and has no significant lethal effect on non-target organisms. It is environmentally friendly and has strong target specificity, which meets the development needs of green agriculture and sustainable agriculture.
[0020] (4) The dual-target fusion dsRNA engineered bacteria preparation method provided by the present invention not only provides an efficient and low-cost solution for the green control of whiteflies, but also provides an important methodological reference for the screening of RNAi targets and the development of RNA biopesticides for other agricultural pests. It has good prospects for industrial application and promotion value. Attached Figure Description
[0021] Figure 1 The lethal effect of spraying bacterial suspensions targeting both the Gawky and CHS genes on adult Q whiteflies in Shouguang population (where control was the blank control group sprayed with water, dsEGFP was the control group sprayed with dsEGFP-expressing bacteria, dsCHS was the control group sprayed with dsCHS-expressing bacteria, dsGawky was the control group sprayed with dsGawky-expressing bacteria, and dsGawky-CHS was the treatment group sprayed with bacteria expressing dual-target fusion dsRNA). Figure 2 The lethal effect of spraying bacterial suspensions targeting both the Gawky and CHS genes on nymphs of the Shouguang population of Q whitefly (where control was the blank control group sprayed with water, dsEGFP was the control group sprayed with dsEGFP-expressing bacteria, dsCHS was the control group sprayed with dsCHS-expressing bacteria, dsGawky was the control group sprayed with dsGawky-expressing bacteria, and dsGawky-CHS was the treatment group sprayed with bacteria expressing dual-target fusion dsRNA). Figure 3The lethal effect of spraying bacterial suspensions targeting both the Gawky and CHS genes on the eggs of the Q whitefly population in Shouguang (where control was the blank control group sprayed with water, dsEGFP was the control group sprayed with bacteria expressing dsEGFP, dsCHS was the control group sprayed with bacteria expressing dsCHS, dsGawky was the control group sprayed with bacteria expressing dsGawky, and dsGawky-CHS was the treatment group sprayed with bacteria expressing dual-target fusion dsRNA). Figure 4 The lethal effect of spraying bacterial suspensions targeting both the Gawky and CHS genes on adult Q whiteflies in the Lingshui population (where control was the blank control group sprayed with water, dsEGFP was the control group sprayed with dsEGFP-expressing bacteria, dsCHS was the control group sprayed with dsCHS-expressing bacteria, dsGawky was the control group sprayed with dsGawky-expressing bacteria, and dsGawky-CHS was the treatment group sprayed with dsRNA-expressing bacteria). Figure 5 The lethal effect of spraying bacterial suspensions targeting both the Gawky and CHS genes on adult whiteflies of type B (where control was the blank control group sprayed with water, dsEGFP was the control group sprayed with dsEGFP-expressing bacteria, dsCHS was the control group sprayed with dsCHS-expressing bacteria, dsGawky was the control group sprayed with dsGawky-expressing bacteria, and dsGawky-CHS was the treatment group sprayed with dsRNA-expressing bacteria). Figure 6 The lethal effect of spraying bacterial solutions with dual targets of the Gawky and CHS genes on adult greenhouse whiteflies (where control is the blank control group sprayed with water, dsEGFP is the control group sprayed with dsEGFP-expressing bacteria, dsCHS is the control group sprayed with dsCHS-expressing bacteria, dsGawky is the control group sprayed with dsGawky-expressing bacteria, and dsGawky-CHS is the treatment group sprayed with bacteria expressing dual-target fusion dsRNA).
[0022] In the above figures, different lowercase letters a, b, c, and d represent significant differences between groups (P<0.05); treatment groups labeled with the same lowercase letter did not have significant differences between groups. Detailed Implementation
[0023] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.
[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.
[0025] The whitefly populations used in the following examples were collected in 2017 from Shouguang, Shandong and Lingshui, Hainan, respectively. These insects were kept on common tobacco variety NC89 in insect-proof cages for an extended period (temperature 27±1 ℃, relative humidity 60%±5%, photoperiod 16L: 8D).
[0026] L4440 plasmid: Wuhan Miaoling Biotechnology Co., Ltd.; HT115(DE3) competent cells: Shanghai Weidi Biotechnology Co., Ltd.
[0027] Example 1
[0028] Preparation of recombinant bacteria with dsRNA fusion of Gawky and CHS genes 1. Total RNA extraction from whiteflies and synthesis of first-strand cDNA.
[0029] Twenty adult whiteflies from the Shouguang population were placed in 1.5 mL centrifuge tubes. Total RNA was extracted from the whiteflies using the TRIzol method. The concentration and quality of RNA were measured using a NanoDropOneC instrument. Reverse transcription was performed using a reverse transcription kit (Evo M-MLV RTMix Kit with gDNA Clean for qPCR, AG) according to the manufacturer's instructions to synthesize the first strand of cDNA.
[0030] 2. Primer design and PCR amplification Based on the gene sequences of the whitefly Gawky (SEQ ID NO.1) and CHS (SEQ ID NO.2), primers for dsRNA were designed and prepared. Using the prepared cDNA as templates, PCR amplification was performed to obtain the Gawky gene amplification product and the CHS gene amplification product. The PCR amplification reaction system was as follows: 25 µL of Premix Taq (TaKaRa Taq™ Version 2.0 plus dye), 2 µL of upstream primer (10 µmol / L), 2 µL of downstream primer (10 µmol / L), 2 µL of cDNA, and ddH2O to a final volume of 50 µL. The reaction program was as follows: 95 °C pre-denaturation for 5 min; denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, 72 °C for 10 s, for 40 cycles; extension at 72 °C for 10 min. After the PCR was completed, the products were recovered.
[0031] 3. Construction of dual-gene target expression vector The L4440 plasmid was double-digested with Sac l and Xho l. Using the OK Clon DNA Ligation Kit Ⅲ Ver.2 (AG), following the multi-fragment cloning procedure in the manufacturer's instructions, the PCR amplification products of the Gawky and CHS genes were simultaneously ligated into the linearized L4440 plasmid. The reaction program was: 50 ℃ for 30 min. 10 μL of the DNA ligation product was transferred into 100 μL of HT115(DE3) competent cells. Finally, 200 μL was plated onto solid medium containing 100 mg / mL Amp+ and incubated overnight at 37 ℃. The next day, six single colonies were randomly selected from the plate, dissolved in medium to prepare a bacterial suspension, and used as a template for PCR reaction using the designed L4440 test primers.
[0032] The PCR amplification product of the Gawky gene was ligated into the linearized L4440 plasmid, transformed into competent cells, and single clones were selected for identification as dsGawky controls.
[0033] The PCR amplification product of the CHS gene was ligated into the linearized L4440 plasmid, transformed into competent cells, and single clones were selected for identification as dsCHS controls.
[0034] Using the green fluorescent protein gene (EGFP, nucleic acid sequence as shown in SEQ ID NO.3) as a control, primers for EGFP gene dsRNA were designed and prepared. Using a plasmid containing the EGFP gene as a template, PCR amplification was performed. After PCR, the product was recovered and ligated into a linearized L4440 plasmid. Competent cells were transformed, and single clones were selected for identification as dsEGFP controls.
[0035] The results showed that the 896bp PCR amplification product obtained by preparing dual-target dsRNAs of the Gawky and CHS genes represented the dsRNA coding regions of the target genes Gawky and CHS. Using a plasmid carrying EGFP as a template, amplification yielded a 507bp PCR product, which represented the EGFP dsRNA coding region. Both target bands were consistent with the sequencing results.
[0036] SEQ ID NO.1 (5'→3'): SEQ ID NO.2(5’→3’): SEQ ID NO. 3 (5'→3'): ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA The primers for preparing dsRNA of Gawky gene and CHS gene are as follows: Forward primer: dsGawky F: 5'-CGAGCTCGGGAGATCCGTCCTACACGCTACA-3' (SEQ ID NO. 4); Reverse primer: dsGawky R: 5'-TTTCTGGACAGTGGGCCCGAGTTGGTGGAGGTTG-3' (SEQ ID NO. 5); Forward primer: dsCHS F: 5'-TCGGGCCCACTGTCCAGAAAGTTTCAAC-3' (SEQ ID NO.6); Reverse primer: dsCHS R:5'-GGTACCGGGCCCCCCCTCGAGTGAGCCAATACCATCCTCTCCA-3' (SEQ ID NO.7); The primers for preparing EGFP gene dsRNA are as follows: Forward primer: dsEGFP F:5'-CGAGCTCGAAGTTCAGCGTGTCCGGCGAG-3' (SEQ ID NO.8); Reverse primer: dsEGFP R: 5'-CCTCGAGGTTCACGTTGATGCCGTTCTTC-3' (SEQ ID NO.9); Example 2
[0037] A bacterial culture for RNAi interference in whiteflies is prepared as follows: The bacteria identified correctly in Example 1 were inoculated into 15 mL of LB medium (Amp+) and cultured at 37 °C with shaking at 200 r / min. When the OD600 value of the bacterial solution reached 0.6, IPTG solution with a final concentration of 0.5 mM was added for induction for 4 h.
[0038] The bacterial culture was centrifuged at 4 ℃ and 5000×g for 10 min to separate the bacterial cells. The supernatant was discarded, and the bacterial cells were resuspended in a small amount of PBS solution. After mixing, the culture was centrifuged at 4 ℃ and 5000×g for 5 min. The supernatant was discarded, and the process was repeated twice to obtain the dsRNA bacterial culture containing the dual gene target sites of the Gawky and CHS genes (denoted as dsGawky-CHS). The bacterial cells were stored in a 20 ℃ refrigerator for later use.
[0039] Example 3
[0040] The lethal effect of spraying a bacterial solution containing a dual-gene target fusion of the Gawky and CHS genes on adult Q whiteflies. An experiment was conducted on the effect of dual-gene target bacterial suspension on the mortality rate of adult whiteflies in the Shouguang (SG) population.
[0041] The bacterial cells prepared in Example 2 were dissolved and diluted with ddH2O to a concentration of 4 g / L (wet cells). The solution was then sprayed onto cotton plants, three times on each side of the leaves. After the solution dried, 30 adult Q whiteflies were released onto the cotton plants, with three replicates per treatment. The same spraying was performed using dsEGFP, dsCHS, and dsGawky as controls. Adult mortality was recorded 5 days after spraying.
[0042] The results are as follows Figure 1 As shown, the mortality rate of adults sprayed with dual-gene target fusion dsRNA bacterial solution was significantly increased compared with the single-target control (dsCHS, dsGawky).
[0043] Example 4
[0044] The lethal effect of spraying a bacterial solution containing a dual-gene target fusion of the Gawky and CHS genes on nymphs and eggs of the Q whitefly. An experiment was conducted in the Shouguang (SG) population to investigate the effects of dual-gene target bacterial suspension on the mortality rates of whitefly nymphs and eggs.
[0045] The bacterial cells prepared in Example 2 were dissolved and diluted with ddH2O to a concentration of 4 g / L (wet cells). Fifteen pairs of whiteflies were released onto cotton plants. On day 9, the number of nymphs was observed under a microscope. The diluted bacterial solution was sprayed onto the cotton plants, with three sprays on each side of the cotton leaves. The same spraying was performed with dsEGFP, dsCHS, and dsGawky as controls. Each treatment was repeated three times. Because the emergence time of whitefly nymphs varied slightly, some nymphs had already emerged as adults by day 17. Since it was not possible to accurately determine the death of nymphs under a microscope, the newly emerged adults were sucked out and their number was recorded every day on day 17. By day 25, there had been no newly emerged adults for three consecutive days. At this point, the nymphs that had not emerged were considered dead.
[0046] Fifteen pairs of whiteflies were released onto cotton plants. After one day of egg-laying, the adults were removed, and the number of eggs on each cotton plant was recorded under a microscope. The diluted bacterial solution prepared in Example 2 was sprayed onto the cotton plants, with three sprays on each side of the cotton leaves. The same spraying was performed with dsEGFP, dsCHS, and dsGawky as controls. Each treatment was repeated three times. Because the hatching time from whitefly eggs to nymphs varies slightly, and whitefly eggs are small, it is not possible to effectively determine whether they are dead under a microscope. On day 10, eggs that did not hatch into nymphs were considered dead.
[0047] The results are as follows Figure 2 and Figure 3 As shown, the mortality rate of nymphs and eggs sprayed with dual-gene target fusion dsRNA bacterial solution was significantly increased compared with the single-target control (dsCHS, dsGawky).
[0048] Example 5
[0049] Effects of spraying a bacterial solution containing a dual-gene target fusion of the Gawky and CHS genes on the Lingshui population of Q-type whitefly, B-type whitefly, and greenhouse whitefly. The bacterial cells prepared in Example 2 were dissolved and diluted using ddH2O to a concentration of 4 g / L (wet cells). The solution was then sprayed onto cotton plants, three times on each side of the leaves. After the solution dried, 30 adult whiteflies of type B (Tobacco Whitefly) or Greenhouse Whitefly were released onto the cotton plants, with three replicates for each treatment. The same spraying was performed using dsEGFP, dsCHS, and dsGawky as controls. The adult mortality rate was recorded 5 days after spraying. Figure 4 , Figure 5 and Figure 6 As shown, spraying dsRNA on the Lingshui population of type Q whitefly, type B whitefly, and greenhouse whitefly resulted in significant differences in mortality rates between the Lingshui population of type Q whitefly and type B whitefly compared to the control group. Figure 4 , Figure 5 Furthermore, the mortality rate of bacteria sprayed with dual-gene target fusion dsRNA was significantly increased compared to the single-target control (dsCHS, dsGawky), while the mortality rate of greenhouse whiteflies showed no significant difference. Figure 6 ).
[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.
Claims
1. The application of dsRNA targeting two genes, Gawky and CHS, in the biological control of whiteflies, characterized in that... The nucleic acid sequence of the Gawky gene is shown in SEQ ID NO.1, and the nucleic acid sequence of the CHS gene is shown in SEQ ID NO.
2.
2. The application according to claim 1, characterized in that, By interfering with the Gawky and CHS genes in whiteflies via RNA interference, the mortality rate of whiteflies is increased.
3. The application according to claim 2, characterized in that, The whitefly mentioned is either type Q or type B.
4. A method for controlling whiteflies, characterized in that, The whitefly's Gawky and CHS genes are interfered with via RNA interference, thereby increasing the whitefly mortality rate. The nucleic acid sequence of the Gawky gene is shown in SEQ ID NO.1, and the nucleic acid sequence of the CHS gene is shown in SEQ ID NO.
2.
5. The method for controlling whiteflies according to claim 4, characterized in that, The specific steps are as follows: (1) Prepare a recombinant vector expressing dual-target dsRNA of the whitefly Gawky gene and CHS gene, and transform it into the expression strain to induce expression; (2) The bacterial cells that induce the expression of dsRNA were prepared into a bacterial solution and sprayed on plants, so that whiteflies would feed on the sprayed plants, thereby increasing the mortality rate of whiteflies.
6. The method for controlling whiteflies according to claim 5, characterized in that, The recombinant vector expressing the dual-target fusion dsRNA of the whitefly Gawky gene and CHS gene was prepared by the following method: Primers for preparing dsRNA of Gawky and CHS genes were used, and cDNA reverse transcribed from total RNA of whiteflies was used as a template for PCR amplification. The amplified fragment was ligated into an expression vector to obtain a recombinant vector expressing dsRNA of dual gene target sites of whiteflies Gawky and CHS genes. The primers for preparing Gawky gene dsRNA are shown in SEQ ID NO.4 and SEQ ID NO.5; The primers for preparing CHS gene dsRNA are shown in SEQ ID NO.6 and SEQ ID NO.
7.
7. The method for controlling whiteflies according to any one of claims 4 to 6, characterized in that, The whitefly mentioned is either type Q or type B.
8. A preparation for controlling whiteflies, characterized in that, A recombinant bacterium containing a dsRNA fusion of the Gawky gene and the CHS gene from the whitefly, wherein the nucleic acid sequence of the Gawky gene is shown in SEQ ID NO.1 and the nucleic acid sequence of the CHS gene is shown in SEQ ID NO.
2.
9. The preparation for controlling whiteflies according to claim 8, characterized in that, The preparation method of the formulation for controlling whiteflies is as follows: (1) Using primers for preparing Gawky and CHS gene dsRNA, and cDNA reverse transcribed from total RNA of whitefly as template, PCR amplification was performed; the amplified fragment was ligated into the expression vector to obtain a recombinant vector expressing dual target dsRNA of Gawky and CHS genes of whitefly. (2) Transform the recombinant vector expressing dual-target dsRNA of the whitefly Gawky gene and CHS gene into the expression strain to induce expression; (3) Centrifuge the bacterial culture after induction to separate the bacterial cells, discard the supernatant, resuspend the bacterial cells in PBS solution, mix well, centrifuge, discard the supernatant, repeat the PBS washing step twice to obtain the preparation for the control of whiteflies.
10. The preparation for controlling whiteflies according to claim 9, characterized in that, The primers for preparing Gawky gene dsRNA are shown in SEQ ID NO.4 and SEQ ID NO.5; the primers for preparing CHS gene dsRNA are shown in SEQ ID NO.6 and SEQ ID NO.7.