Application of GST gene and its siRNA combined with different biocontrol bacteria in termite control

Abstract

The invention discloses a black-chested termite GST The application of genes and their siRNA combined with different biocontrol bacteria (Metarhizium anisopliae or Beauveria bassiana) in termite control, GST siRNA from Reticulitermes nigrotermes GST Gene, namely glutathione S-transferase gene. Cloning target gene GST The fragment, sequence is SEQ 1. Design and cloning GST dsRNA template, synthesized by T7 in vitro transcription system GST dsRNA. GST Digestion of dsRNA into more potent GST siRNA. GST siRNA is mixed with termite food and placed in a termite colony for the termites to feed on. GST siRNA in termites GST Gene expression decreased significantly. GST After the termites were treated with siRNA, the mortality rate of termites infected with fungi increased significantly; GST After the termites were injected with siRNA, the mortality rate of infected termites increased significantly.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to GST Genes and GST siRNA, a double-stranded nucleic acid that enhances the effectiveness of biocontrol bacteria in killing termites GST siRNA. Background Art

[0002] Termites are a kind of pests that are ubiquitous worldwide, especially in tropical and subtropical regions. Their social lifestyle has caused irreversible damage to houses, reservoirs, dams, mountainous areas, rural areas, communication equipment, etc., and this damage is highly hidden and serious. At present, chemical control methods are the main means of termite control. However, many highly effective but highly toxic and highly polluting chemical agents have been banned, such as chlordane and mirex. Therefore, based on considerations for the safety of the environment and humans and animals, how to control termites in a green way is one of the key issues that researchers are currently trying to solve.

[0003] Biological pest control refers to the control technology of pest population by using beneficial organisms and their products. It is the most successful, economical and safest green control technology. In nature, insects killed by fungi account for about 60% of all deaths caused by pathogenic microorganisms. At present, the main ones that have been used in production are Metarhizium anisopliae, Beauveria bassiana, Penicillium pseudomycota, and Nomurium rapae. For example, Metarhizium rapae belongs to the subphylum Ascomycota, class Pyromycetes, order Glomerales, family Claviceps, and genus Metarhizium. Its biological preparations can control more than 200 kinds of agricultural pests and can form a rhizosphere symbiotic relationship with plant roots, thereby continuously controlling harmful insects. Another completely different entomogenous fungus, Beauveria bassiana, belongs to the subphylum Peziomycota, class Felomycetes, order Hypocreales, family Cordyceps, and genus Beauveria. It can invade more than 200 kinds of insects and mites in 15 families of 6 orders and is the most widely used in the world. Therefore, biological control fungi play an important role in the continuous green control of harmful insects. However, the effect of using biocontrol bacteria to control termite colonies in the wild is far from ideal. This is because termites are social insects, and their nest members use behavioral immunity and physiological immunity to cooperate with each other to resist the infection of pathogenic fungi. Therefore, how to weaken the disease defense of termites is the key to improving the effectiveness of biocontrol bacteria in killing termites.

[0004] RNA interference (RNAi) refers to the phenomenon of highly conserved, double-stranded RNA (dsRNA)-induced, efficient and specific degradation of homologous mRNA in the process of evolution. Insect RNAi has strong specificity, is environmentally friendly, and has high efficiency. It has been widely used in gene function, pesticide resistance research, and pest control. For example, inhibiting the gene expression of chitin synthase A by feeding related dsRNA can significantly hinder the development of beet armyworm larvae and cause the death of larvae; inhibiting the gene expression of acyl-CoA reductase by feeding related dsRNA can effectively inhibit the development of black stink bugs and achieve the purpose of killing. In termites, dsRNA is introduced through microinjection technology to inhibit the expression of isocitrate dehydrogenase, which in turn leads to an increase in the mortality rate of termites infected with green muscardine, but this method is too complicated to operate; inhibiting the growth of termites by feeding high doses of dsRNA (total feeding amount of about 100 μg) is not suitable for termites. Dicer-1 The gene expression of termites infected with green muscardine is inhibited, which leads to an increase in the mortality of termites infected with green muscardine. However, this method requires too high a dsRNA dosage. These problems seriously limit the application of RNAi in biological control of termites. Therefore, this patent aims to apply for a low-dose, high-efficiency double-stranded nucleic acid that can be fed to weaken the physiological defense of termites, thereby increasing the killing effect of biocontrol bacteria.

[0005] During the process of infecting the host, pathogens often secrete toxic substances that damage the host's immune, metabolic, and nervous systems, thereby facilitating their own colonization in the host. GST ) plays an important role in the physiological defense of animals, mainly participating in the decomposition and metabolism of endogenous and exogenous toxic substances, and plays a very important role in the detoxification process of insects. GST Gene expression was significantly increased, indicating GST It plays an important role in the process of termite decomposition of pathogenic bacteria toxins. GST As the target gene, the detoxification effect of GST in termites is inhibited to improve the killing effect of biocontrol bacteria on termites. Compared with dsRNA, the small molecule product siRNA after cleavage has a higher interference efficiency. Therefore, the present invention intends to use GST siRNA instead GST dsRNA for termite RNAi. In addition, some nanoparticles, as nucleic acid drug carriers, have broad application prospects in improving the performance of nucleic acid drugs (improving cell permeability, protecting drug stability, controlling drug release and prolonging drug efficacy, etc.). Therefore, the present invention also intends to feed GST The siRNA nanocomplex improves the killing effect of biological control bacteria on termites. The invention is beneficial to promoting the development of RNAi-mediated termite biological control technology. Summary of the invention

[0006] The purpose of the present invention is to provide a target gene GST as well as GST Application of siRNA in biological control of termites: Improving the insecticidal effect of biocontrol bacteria by weakening the detoxification ability of termites.

[0007] The present invention GST The siRNA was designed from a key detoxification gene in termites, glutathione S-transferase ( GST ), the DNA fragment sequence is derived from the transcriptome database of Reticulitermes nigromaculata, SEQ ID NO: 1, with a length of 703 bp.

[0008] By designing upstream primer SEQ ID NO: 2 and downstream primer SEQ ID NO: 3, PCR amplification, TA cloning or blunt-end cloning, bacterial picking detection and plasmid recovery were performed using termite cDNA as a template to obtain a plasmid containing the target gene. GST Purified plasmid fragment.

[0009] Specific primers containing "transcription enhancer" and "T7 promoter" were designed according to SEQ ID NO: 5 for the upstream primer and 6 for the downstream primer to contain the target gene. GST The fragment was amplified by PCR using the pure plasmid as a template, purified by phenol / chloroform / isoamyl alcohol (25:24:1) reagent, and concentrated by sodium acetate combined with anhydrous ethanol. GST dsRNA template, template length 557 bp, DNA sequence is shown in SEQ ID NO:4.

[0010] GST The dsRNA template was synthesized by T7 in vitro transcription system, purified by phenol / chloroform (1:1) reagent, and concentrated by sodium acetate combined with anhydrous ethanol to obtain double-stranded nucleic acid. GST dsRNA, product length 505 bp, RNA sequence is shown in SEQ ID NO:7.

[0011] Double-stranded nucleic acid GST dsRNA is digested by RNase III to obtain small double-stranded nucleic acids GST siRNA.

[0012] Based on the above gene sequence and method, the present invention also provides a small molecule double-stranded nucleic acid GST siRNA products for controlling termites.

[0013] A small double-stranded nucleic acid GST A product for controlling termites with siRNA as the active ingredient.

[0014] Small double-stranded nucleic acid GST The product for preventing and controlling termites is prepared by siRNA in collaboration with biocontrol fungi, wherein the biocontrol fungi include Metarhizium anisopliae, Metarhizium luteum, Metarhizium locust and its closely related Metarhizium anisopliae, Beauveria bassiana, Beauveria bassiana microsporus, Beauveria bassiana and its closely related Beauveria bassiana.

[0015] A method for preventing and controlling termites, comprising the small molecule double-stranded nucleic acid GST siRNA, the steps are as follows:

[0016] (1) GST siRNA is mixed with termite food and placed in a termite colony for termites to eat. GST siRNA termites;

[0017] (2) GST siRNA is first mixed with nanoparticles to form GST The siRNA nanocomplex is then mixed with termite food and placed in a termite colony for termites to eat. GST Termites with siRNA nanocomplexes;

[0018] (3) using a 0.1-2% Tween 80 solution to collect biocontrol fungal spores and prepare a biocontrol fungal spore suspension;

[0019] (4) Using a suspension of biocontrol fungi spores to infect and feed on insects GST siRNA for termites, thereby preventing and controlling termites.

[0020] Termite food includes cellulose-containing materials such as paper, wood chips, sawdust, or wood powder. GST siRNA or GST The siRNA nanocomplex is mixed with termite food by a mixing method of infiltration, coating, or injection.

[0021] Nanoparticles include chitosan, layered double hydroxides, carbon quantum dots, Lipofectamine 3000, Entranster TM -in vivo.

[0022] The biocontrol bacteria include Metarhizium anisopliae, Metarhizium luteum, Metarhizium locust and its closely related Metarhizium anisopliae, Beauveria bassiana, Beauveria bassiana microspores, Beauveria bassiana and its closely related Beauveria bassiana.

[0023] The present invention synthesizes the small molecule double-stranded nucleic acid GST Monitoring the interference effect of siRNA on black-chested termites: GST siRNA or GFPThe filter paper is soaked with the siRNA solution and then placed in a petri dish for termites to feed on. GST siRNA-treated termites were fed with the same amount of GFP siRNA-treated termites were used as the control group. RT-qPCR showed that compared with the control group, termites fed for 1 day GST siRNA treatment group termites GST Gene expression was significantly reduced. GST The enzyme activity kit test showed that compared with the control group, the GST siRNA treatment group termites GST The enzyme activity was significantly reduced. GST siRNA can significantly inhibit GST Gene expression and GST enzyme activity.

[0024] The present invention provides GST Application of siRNA in synergistic biocontrol bacteria in termite biological control: GST The filter paper was soaked with siRNA solution and then placed in a culture dish for termites to feed. After feeding for 1 day, the spore suspension of Metarhizium anisopliae or Beauveria bassiana was sprayed on the body surface of the termites as the treatment group; GFP The filter paper was soaked with siRNA solution and then placed in a culture dish for termites to feed. After feeding for 1 day, the spore suspension of Metarhizium anisopliae or Beauveria bassiana was sprayed on the body surface of the termites as a control group. The number of deaths of infected termites in the above-mentioned treatment and control groups was counted every day for 10 days. The results showed that the mortality rate of infected termites in the treatment group was significantly higher than that of infected termites in the control group. This shows that GST siRNA can significantly increase the mortality rate of termite infection and enhance the control effect of biocontrol bacteria on termites.

[0025] The present invention provides GST Application of siRNA nanocomplexes in synergistic biocontrol bacteria in termite biological control: GST siRNA and GFP siRNA and Entranster TM -in vivo mixed to form a "siRNA-nano" complex. GST The filter paper was soaked with the siRNA nanocomplex solution and placed in a culture dish for termites to feed. After feeding for 1 day, the termites were sprayed with a suspension of Metarhizium anisopliae or Beauveria bassiana spores as the treatment group. GFPThe filter paper was soaked with the siRNA nanocomplex solution and then placed in a culture dish for termites to feed. After feeding for 1 day, the termites were sprayed with a suspension of Metarhizium anisopliae or Beauveria bassiana spores as a control group. The number of dead termites infected in the treatment and control groups was recorded every day for a total of 10 days. The results showed that the mortality rate of termites infected in the treatment group was significantly higher than that in the control group. This shows that GST siRNA nanocomplexes can increase termite infection mortality and significantly improve the control effect of biocontrol bacteria on termites.

[0026] The present invention has the following four obvious advantages: 1. GST 1. siRNA is a double-stranded RNA with a simple preparation method, strong specific targeting, and is environmentally friendly. It can weaken the detoxification ability of termites through a simple feeding operation; 2. It can cooperate with two different types of biocontrol bacteria (Metarhizium and Beauveria bassiana) to kill termites with ideal results; 3. Compared with dsRNA in traditional RNAi methods, siRNA has higher interference efficiency, less dosage, and higher gene silencing effect; 4. GST siRNA and its nanocomplex can be used to perform termite RNAi by feeding, which is easier to operate and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : GST dsRNA and GST siRNA, GFP dsRNA and GFP Gel electrophoresis of siRNA. Band 1 is GFP dsRNA; Band 2 is GFP siRNA; Band 3 is GST dsRNA; Band 4 is GST siRNA.

[0028] Figure 2 : Termites took 20 μg GST dsRNA and 20 μg GST siRNA 1 day later GST Changes in gene expression. GST dsRNA or GST siRNA-treated termites were fed GFP dsRNA or GFP siRNA termites were the control group; as shown in the figure, compared with the respective control groups, GST dsRNA in termites GST There was no significant change in gene expression, but the same amount of food was consumed GST siRNA in termites GSTGene expression was significantly reduced; the bar graph is the mean ± standard error, * indicates P <0.05; ns indicates no significant difference.

[0029] Figure 3 :Termite feeding 20 μg GST siRNA 1 day later GST Changes in enzyme activity. GST siRNA-treated termites were fed GFP siRNA termites were the control group; as shown in the figure, compared with the control group, GST siRNA in termites GST The enzyme activity was significantly reduced; the bar graph is the mean ± standard error, * indicates P <0.05.

[0030] Figure 4 : Termite feeding GST Death effect diagram after siRNA infection. A. Death effect diagram of Metarhizium anisopliae infection; B. Death effect diagram of Beauveria bassiana infection; Feeding GST siRNA-treated termites were fed GFP siRNA termites were the control group; as shown in the figure, compared with the control group, GST The mortality rate of termites infected with siRNA increased significantly; ** indicates P <0.01.

[0031] Figure 5 : GST The control effect of siRNA combined with biocontrol bacteria. A. Metarhizium anisopliae; B. Dead termites infected with Metarhizium anisopliae; C. Beauveria bassiana; D. Dead termites infected with Beauveria bassiana.

[0032] Figure 6 : Termite feeding GST The death effect of siRNA nanocomplex infection. A. Death effect of Metarhizium anisopliae infection; B. Death effect of Beauveria bassiana infection; Termites feed on GST siRNA nanocomplex obtains the main component GST siRNA, as the treatment group, termites fed GFP siRNA nanocomplex obtains the main component GFP siRNA, as the control group; as shown in the figure, compared with the control group, GST The mortality rate of termites infected with the main component of siRNA increased significantly; ** indicates P <0.01. * indicates P <0.05. DETAILED DESCRIPTION

[0033] Embodiment 1: GST siRNA preparation process

[0034] 1. Extract total RNA from Reticulitermes nigrotermes and obtain termite cDNA library by reverse transcription.

[0035] 2. Using termite cDNA as template, GST Gene-specific primers SEQ ID NO: 2 (upstream primer: 5'-CTGTGC GAA GAC ACC ACT GA-3') and SEQ ID NO: 3 (downstream primer: 5'-TAT GTC TCA TGC AAA CCCCT-3') were obtained by PCR amplification. GST The gene fragments were then separated and purified by agarose gel electrophoresis. GST The gene fragment has a DNA sequence of SEQ ID NO: 1.

[0036] 3. Through TA cloning GST The gene fragment was inserted into the PMD-18T plasmid, and then the target gene GST The plasmid of the fragment was transformed into DH5α competent cells and cultured in a shaker at 37°C and 200 rpm for 1 h.

[0037] 4. Take the competent cell culture liquid after the above transformation, spread it evenly in the ampicillin resistance medium, and culture it at 37℃ for 24h.

[0038] 5. Pick a single plaque and place it in a new ampicillin-resistant culture medium, incubate at 37°C for 4 h, then perform bacterial solution PCR, perform gel electrophoresis, and finally select the plaque containing the target gene. GST The bacterial solution of the fragment is subjected to PCR and expanded culture.

[0039] 6. Centrifuge the cultured bacterial solution at 25℃ 4000 rpm, concentrate it, and then use a plasmid recovery kit to extract the plasmid to obtain the target gene. GST The plasmid containing the target gene was obtained and stored at -20℃. GST Purified plasmid fragment.

[0040] 4. Using the above plasmid as template, use the plasmid containing "transcription enhancer" and "T7 promoter" GSTThe fragment-specific primers SEQ ID NO: 5 (upstream primer: 5'-GAT CAC TAA TAC GAC TCA CTA TAG GGC TGT GCG AAGACA CCA CT-3') and SEQ ID NO: 6 (downstream primer: 5'-GAT CAC TAA TAC GAC TCA CTA TAG GGGACA AGT CAG CAA CTG TA-3') were amplified by PCR. GST dsRNA template. Collection GST dsRNA template PCR reaction system, add water to 300 μL. Then add 30 μL of sodium acetate solution and 300 μL of phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) solution, centrifuge at 13200 rpm for 15 min, and take the supernatant. Add 2 times the volume of anhydrous ethanol, after -20℃ overnight, centrifuge at 13200 rpm for 10 min, and discard the supernatant. Finally, wash the precipitate with 75% alcohol, dry, and dissolve the precipitate, thereby obtaining a high concentration of GST dsRNA template.

[0041] 5. Utilize the above GST Obtaining dsRNA template and T7 in vitro transcription system GST dsRNA. Collect the reaction system and add enzyme-free water to 300 μL. Then add 30 μL of sodium acetate, 150 μL of water-saturated phenol and 150 μL of chloroform (volume ratio 1:1), centrifuge at 13200 rpm for 15 min, and take the supernatant. Add 2 times the volume of anhydrous ethanol, after overnight at -20℃, centrifuge at 13200 rpm for 10 minutes, and discard the supernatant. Finally, wash the precipitate with 75% alcohol, dry, and dissolve the precipitate to obtain GST dsRNA.

[0042] 6. Digestion with RNase III GST dsRNA GST siRNA( Figure 1 ).

[0043] Example 2: Detection GST Reticulitermes chinensis GST Genetic influence

[0044] 20 μg of the above GST dsRNA or GST siRNA was used to wet a 1.5 cm diameter circular filter paper and placed in a 3.5 cm diameter petri dish for 9 black-chested termites to eat as the treatment group. GFP dsRNA or GFP1.5 cm diameter circular filter paper was moistened with siRNA and placed in a 3.5 cm diameter petri dish for feeding by 9 black-chested termites as a control. After feeding for 1 day, total RNA of termites was extracted and RT-qPCR was used to detect the expression of RNA in termites with different treatments. GST The experiment was repeated 6 times, and the Wilcoxon test was used to analyze the differences. Figure 2 As shown, compared with the respective control groups, GST dsRNA treated termites GST There was no significant change in gene expression ( Figure 2 A, P = 0.463), but GST siRNA treated termites GST Gene expression was significantly reduced by 58.8% ( Figure 2 B, P<0.05), indicating GST siRNA can better inhibit the GST Gene expression.

[0045] Example 3: Detection GST Effects of siRNA on the detoxification of GST in Reticulitermes nigrotermes

[0046] 20 μg of the above GST siRNA was used to wet a 1.5 cm diameter circular filter paper, which was placed in a 3.5 cm diameter petri dish and fed to nine black-chested termites for 1 day as the treatment group. GST siRNA was used to wet a 1.5 cm diameter circular filter paper and placed in a 3.5 cm diameter culture dish for 9 black-chested termites to feed on for 1 day as a control group. Termite tissue fluid was extracted and stored in a -20°C refrigerator for later use. The GST enzyme activity in termites of different treatment groups was detected using a GST enzyme activity detection kit. The experiment was repeated 6 times and the differences were analyzed using a paired T test. The results are shown in Figure 3 As shown in the figure, compared with the control group, the GST enzyme activity in the treated termites was significantly reduced by 34.4% (P<0.01), indicating that GST siRNA can inhibit the detoxification of infected termites.

[0047] Example 4: Detection GST Effect of siRNA on the ant-killing effect of Metarhizium anisopliae

[0048] The M. anisopliae was cultured in potato dextrose medium (PDA). After 2 weeks of culture, the spores of M. anisopliae were collected with 1% Tween 80 solution, and a spore suspension was prepared and stored in a refrigerator at 4°C for later use. The concentration of the spore suspension was 10 6 spores / mL.

[0049] 20 μg of the aboveGST 1.5 cm diameter circular filter paper was moistened with siRNA and placed in a 3.5 cm diameter petri dish for 9 black-chested termites to feed on for 1 day. Then, the above-mentioned Metarhizium spore suspension was sprayed on the body surface of black-chested termites as the treatment group. GFP siRNA was used to wet a 1.5 cm diameter circular filter paper, which was placed in a 3.5 cm diameter petri dish and fed to 9 black-chested termites for 1 day. Then, an equal amount of Metarhizium spore suspension was sprayed on the body surface of black-chested termites as a control group. The number of dead termites infected with fungi was recorded every day and the dead individuals were removed in time. The experiment was repeated 4 times, and the Kaplan-Meier method was used to analyze the data differences. The results are shown in Figure 2. Figure 4 A and Figure 5 As shown in A and B, the mortality rate of termites infected with fungi in the control group [LT 50 : 7.936 (7.241-8.873)], compared with the treatment group, the mortality rate of termites infected with fungi [LT 50 : 4.299 (3.180-5.170)] increased significantly ( χ 2 =26.637, P<0.01), indicating GST siRNA can significantly improve the effectiveness of Metarhizium anisopliae in killing termites.

[0050] Example 5: Detection GST Effect of siRNA on the ant-killing effect of Beauveria bassiana

[0051] The method and steps are the same as those in Example 4, except that Metarhizium anisopliae is replaced by Beauveria bassiana. The results are as follows: Figure 4 As shown in B, the mortality rate of termites infected with fungi in the control group [LT 50 : 6.947 (6.434-7.495)], compared with the treatment group, the mortality rate of termites infected with fungi [LT 50 : 3.840 (3.464-4.180)] increased significantly ( χ 2 = 29.371, P < 0.01), indicating GST siRNA can significantly improve the effectiveness of Beauveria bassiana in killing termites ( Figure 4 B and Figure 5 C, D).

[0052] Example 6: Detection GST Effect of siRNA nanocomplex on the ant-killing effect of Metarhizium anisopliae

[0053] Use 1% Tween 80 solution to collect the spores of Metarhizium anisopliae, prepare a spore suspension, and store it in a refrigerator at 4°C for later use. 6 spores / mL.

[0054] Using Entranster TM -in vivo nanotransfection kit, refer to the instructions and make slight modifications, first dilute 10 μL of nanoparticle stock solution to 40 μL with enzyme-free water, then dilute 20 μg GST siRNA or GFP siRNA to 30 μL, and finally mix the two dilutions, immediately vortex for 30 s, and stand at room temperature for 10 min to prepare GST siRNA or GFP The siRNA nanocomplex is ready for use.

[0055] 20 μg of the above GST The siRNA nanocomplex was used to wet a 1.5 cm diameter circular filter paper, which was placed in a 3.5 cm diameter culture dish and fed to nine black-chested Reticulotermes for 1 day. Subsequently, the above-mentioned Metarhizium spore suspension was sprayed on the body surface of black-chested Reticulotermes as the treatment group. GFP The siRNA nanocomplex was used to wet a 1.5 cm diameter circular filter paper and placed in a 3.5 cm diameter petri dish for 9 black-chested termites to feed on for 1 day. Then, an equal amount of Metarhizium spore suspension was sprayed on the body surface of black-chested termites as a control group. The number of dead termites infected with fungi was recorded every day and the dead individuals were removed in time. The experiment was repeated 4 times, and the Kaplan-Meier method was used to analyze the data differences. The results are shown in Figure 2. Figure 6 As shown in A, compared with the control group infected termites [LT 50 : 4.828 (4.192-5.346)], the mortality rate of termites infected with fungi in the treatment group [LT 50 : 3.551 (0.864-5.144)] increased significantly ( χ 2 =12.429, P<0.01), indicating GST The siRNA nanocomplex can significantly improve the effectiveness of biocontrol bacteria in killing termites.

[0056] Example 7: Detection GST Effects of siRNA nanocomplex on the killing effect of Beauveria bassiana on ants

[0057] The method and steps are the same as those in Example 6, except that the Metarhizium anisopliae is replaced by Beauveria bassiana. The results are as follows: Figure 6 As shown in B, the mortality rate of termites infected with fungi in the control group [LT 50 : 5.106 (4.515-5.601)], compared with the control group, the mortality rate of termites infected with fungi [LT 50 : 3.991 (3.557-4.389)] increased significantly (χ 2 = 4.640, P<0.05), indicating GST The siRNA nanocomplex can significantly enhance the effectiveness of Beauveria bassiana in killing termites.

Claims

1. Black-chested Reticulitermes GST Gene, It is characterized in that The DNA sequence is SEQ ID NO:

1.

2. A small molecule double-stranded nucleic acid GST siRNA, It is characterized in that Using the black-chested termite described in claim 1 GST The plasmid of the gene fragment was used as a template, the upstream primer was SEQ ID NO: 5, the downstream primer was SEQ ID NO: 6, and the PCR amplification, phenol / chloroform / isoamyl alcohol reagent purification, sodium acetate combined with anhydrous ethanol concentration method was used to obtain GST dsRNA template, GST The DNA sequence of the dsRNA template is SEQ ID NO: 4; GST The dsRNA template was synthesized by T7 in vitro transcription system, purified by phenol / chloroform reagent, and concentrated by sodium acetate combined with anhydrous ethanol to obtain double-stranded nucleic acid. GST dsRNA, RNA sequence is SEQ ID NO: 7; Double-stranded nucleic acid GST dsRNA is digested by RNase III to obtain small double-stranded nucleic acids GST siRNA.

3. The small molecule double-stranded nucleic acid according to claim 2 GST siRNA, It is characterized in that Reticulitermes nigrotermes GST Reticulitermes nigrotermes GST The gene was used as a template, and the upstream primer SEQ ID NO: 2 and the downstream primer SEQ ID NO: 3 were used to obtain the plasmid through PCR amplification, TA cloning, shake detection, and plasmid extraction.

4. A small molecule double-stranded nucleic acid GST siRNA termite control product, the small molecule double-stranded nucleic acid GST siRNA is a small molecule double-stranded nucleic acid as described in any one of claims 2-3 GST siRNA.

5. A double-stranded nucleic acid containing the small molecule according to claim 4 GST A product for controlling termites with siRNA as the active ingredient.

6. The product according to claim 5, It is characterized in that Double-stranded nucleic acid GST The termite control product is prepared by siRNA in cooperation with biocontrol fungi, and the biocontrol fungi include Metarhizium anisopliae, Metarhizium luteum, Metarhizium locust, Beauveria bassiana, Beauveria bassiana microsporus or Beauveria bassiana.

7. A method for controlling black-chested termites, comprising using the small molecule double-stranded nucleic acid according to any one of claims 2 to 3 GST siRNA, It is characterized in that Here are the steps: (1) Small molecule double-stranded nucleic acid GST siRNA is mixed with termite food and placed in a termite colony for termites to eat. GST siRNA termites; (2) Small molecule double-stranded nucleic acid GST siRNA is first mixed with nanoparticles to form GST The siRNA nanocomplex is then mixed with termite food and placed in a termite colony for termites to eat. GST Termites with siRNA nanocomplexes; (3) collecting biocontrol fungal spores using a 0.1-2% Tween 80 solution to prepare a biocontrol fungal spore suspension; (4) Using a suspension of biocontrol fungi spores to infect and feed on insects GST siRNA or GST siRNA nanocomplexes are used to treat termites, thereby preventing and controlling termites.

8. The method for controlling black-chested Reticulitermes according to claim 7, It is characterized in that Termite food includes cellulose-containing materials such as paper, wood chips, sawdust, or wood powder. GST siRNA or GST The siRNA nanocomplex is mixed with termite food by a mixing method of infiltration, coating, or injection.

9. The method for controlling black-chested Reticulitermes according to claim 7, It is characterized in that Nanoparticles include chitosan, layered double hydroxides, carbon quantum dots, Lipofectamine 3000, Entranster TM -in vivo.

10. The method for controlling Reticulitermes fuscata according to claim 7, It is characterized in that The biocontrol bacteria include Metarhizium anisopliae, Metarhizium luteum, Metarhizium locust, Beauveria bassiana, Beauveria bassiana microspores or Beauveria bassiana.

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