Application of SOD siRNA combined with Metarhizium, Beauveria bassiana or Paecilomyces oxytoca in termite control

Abstract

The invention discloses a double-stranded nucleic acid SOD siRNA, using double-stranded nucleic acid SOD dsRNA was obtained by RNase III digestion SOD siRNA. A double-stranded nucleic acid SOD siRNA termite control products. Then RNase III is used to SOD Digestion of dsRNA into more potent SOD siRNA. SOD siRNA is mixed with termite food and then placed in a termite colony for the termites to eat. SOD After siRNA, in vivo SOD The gene expression level decreased significantly, and the SOD enzyme activity decreased significantly. SOD After the termites were treated with siRNA, the mortality rate of infected termites increased significantly. SOD siRNA can significantly improve the biological control effect of Metarhizium, Beauveria bassiana and Paecilomyces oxysporum on termites.

Description

Technical Field

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

[0002] Termites are a global economic pest that harms garden trees, crops, houses and reservoir dams, posing a huge threat to human life and property. The economic losses caused by termites in my country reach 10 billion yuan each year, so termite control is highly valued. Chemical control is the main means of termite control. However, many highly toxic and highly polluting chemical agents have been banned. Therefore, research and development of new non-toxic, pollution-free and environmentally friendly biological control agents has become one of the urgent needs in the field of termite control.

[0003] In nature, insects killed by fungi account for about 60% of all deaths caused by pathogenic microorganisms. The use of insect-specific pathogenic fungi can effectively control the number of harmful insect populations, and has therefore been widely studied and applied. Metarhizium )、Beauveria bassiana( Beauveria ) and Paecilomyces spp. Paecilomyces ) is the most successful, economical, widespread and safest biocontrol agent in green control technology. Its host insects include harmful insects such as Lepidoptera, Coleoptera, Hymenoptera, Homoptera, Diptera, and Hemiptera. These insect-specific pathogenic fungi can attach to the insect epidermis, germinate at a suitable temperature, produce germ tubes, and form invasive nails. Then they secrete chitinase, dissolve the insect body wall, enter the insect body cavity, and invade the insect's fat tissue and muscle. Finally, they reproduce in the insect body, plunder nutrients, produce toxins, and then cause the insect to die. However, the effect of using entomogenous fungi to control termite nests in the wild is not ideal. This is because termites are social insects, and their nest members rely on the social immune system and have evolved a series of disease defense strategies in behavioral and physiological defense (such as grooming, body surface disinfection, "vaccination", immune response, detoxification and antioxidant response, etc.). Therefore, how to weaken the disease defense of termites is the key to improving the effectiveness of biocontrol bacteria in killing termites.

[0004] In the process of resisting fungal infection and decomposing fungal toxins, termites infected with fungi produce a large number of superoxide anion free radicals, and excessive superoxide anion free radicals can damage cell membranes, denature proteins, and destroy DNA structures. Superoxide dismutase (SOD) is an important antioxidant enzyme widely distributed in microorganisms, plants, and animals. It can catalyze the dismutation of superoxide anion free radicals to generate oxygen and hydrogen peroxide, protecting the body from oxidative damage. Previous studies have shown that in the bodies of termites infected with fungi, superoxide anion free radicals are produced in large quantities, which can damage cell membranes, denature proteins, and destroy DNA structures. SODThe gene expression and SOD enzyme activity increased significantly, indicating that SOD plays an important role in termite resistance to pathogen infection. SOD Gene is the target gene, by feeding termites SOD siRNA silencing SOD Gene, weakening the antioxidant activity of termite SOD enzyme, thereby improving the killing effect of biocontrol bacteria on termites.

[0005] At present, the method of combining RNAi with biocontrol bacteria to synergistically control harmful insects (such as harmful insects such as Lepidoptera, Homoptera and Blattodea) has attracted widespread attention from researchers. However, in termite control, most RNAi pesticides remain on dsRNA with larger molecules and lower efficiency, resulting in high cost and low efficiency of termite RNAi, which restricts the development of termite RNAi pesticides. In addition, almost all research on termite biocontrol bacteria is mainly focused on Metarhizium anisopliae, and there are few studies on RNAi synergistic with other biocontrol bacteria. In view of this, the present invention intends to digest dsRNA into smaller, easier to absorb, and more efficient siRNA based on dsRNA. At the same time, in addition to Metarhizium anisopliae, the present invention also intends to seek breakthroughs in the effects of other biocontrol bacteria. The results show that the present invention SOD siRNA ratio SOD dsRNA has a higher interference efficiency. SOD siRNA has a wide range of synergistic effects, and has synergistic effects on Metarhizium, Beauveria bassiana and Paecilomyces. SOD siRNA combined with Beauveria bassiana has a higher efficiency in killing termites. Summary of the invention

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

[0007] The present invention SOD The siRNA was designed from a key antioxidant gene in termites, the superoxide dismutase gene ( SOD ), the DNA sequence was derived from the black-chested Reticulitermes transcriptome database, which is SEQ 1. The upstream primer SEQ ID NO: 2 and the downstream primer SEQ ID NO: 3 were designed based on SEQ ID NO: 1, and 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. SOD A pure plasmid containing the target gene SOD The fragment length is 648 bp, and the DNA sequence is shown in SEQ ID NO:4.

[0008] Specific primers containing "transcription enhancer" and "T7 promoter" were designed based on SEQ ID NO: 4, the upstream primer was SEQ ID NO: 5, and the downstream primer was SEQ ID NO: 6 to contain the target gene. SOD The purified plasmid of the fragment was used as a template for PCR amplification and concentrated by sodium acetate combined with anhydrous ethanol. SOD dsRNA template, template length 581 bp, DNA sequence is shown in SEQ ID NO:7.

[0009] SOD dsRNA template was obtained by in vitro transcription system SOD dsRNA, concentrated by sodium acetate combined with anhydrous ethanol SOD dsRNA, product length 529 bp, RNA sequence is shown in SEQ ID NO:8.

[0010] SOD dsRNA was obtained by RNase III digestion SOD siRNA.

[0011] Based on the above gene sequence and method, the present invention also provides a double-stranded nucleic acid SOD siRNA products for controlling termites.

[0012] A double-stranded nucleic acid SOD A product for controlling termites with siRNA as the active ingredient.

[0013] Double-stranded nucleic acid SOD The product for preventing and controlling termites is prepared by siRNA in cooperation with biocontrol fungi, wherein the biocontrol fungi include one or more of Metarhizium, Beauveria bassiana or Paecilomyces oxysporum.

[0014] A method for preventing and controlling termites, comprising the double-stranded nucleic acid SOD siRNA, the steps are as follows:

[0015] (1) Double-stranded nucleic acid SOD siRNA is mixed with termite food and placed in a termite colony for termites to eat. SOD siRNA termites;

[0016] (2) using a 0.1-2% Tween 80 solution to collect biocontrol fungal spores and prepare a biocontrol fungal spore suspension;

[0017] (3) Using a suspension of biocontrol fungi spores to infect and feed on insects SOD siRNA for termites, thereby preventing and controlling termites.

[0018] Termite food includes cellulose-containing materials such as paper, wood chips, sawdust, or wood powder. SOD The siRNA is mixed with termite food by dipping, smearing, or injecting.

[0019] The biocontrol bacteria include one or more of Metarhizium, Beauveria bassiana, or Paecilomyces oxysporum.

[0020] The present invention is synthesized by the above SOD Monitoring the interference effect of siRNA on black-chested termites: SOD siRNA or GFP The filter paper was soaked with siRNA solution and then placed in a petri dish for termites to feed on. After feeding for 1 day, RT-qPCR was performed on the termites in the petri dish. SOD siRNA-treated termites were fed with the same amount of GFP The results showed that compared with the control group, termites fed with siRNA for 1 day SOD siRNA treatment group termites SOD The gene expression level was significantly decreased.

[0021] The present invention is synthesized by the above SOD The inhibitory effect of siRNA on enzyme activity in black-chested termites was tested: SOD The filter paper was soaked with siRNA solution and then placed in a culture dish for termites to feed on. After feeding for 1 day, the filter paper was used as the treatment group. GFP The filter paper was soaked with siRNA solution and then placed in a petri dish for termites to feed. After feeding for 1 day, it was used as the control group. It was ground in liquid nitrogen and centrifuged to obtain the supernatant. The SOD enzyme activity kit was used to detect that compared with the control group, the feeding SOD The activity of SOD enzyme in termites infected with siRNA was significantly reduced.

[0022] The present invention is synthesized by the above SOD The synergistic effect of siRNA on three biocontrol bacteria was tested: SOD The filter paper was soaked with siRNA solution and then placed in a culture dish for termites to feed on. After feeding for 1 day, the filter paper was used as the treatment group. GFP The filter paper was soaked with siRNA solution and then placed in a culture dish for termites to feed on. After feeding for 1 day, it was used as the control group. The termites in the treatment group and the control group were infected with Metarhizium anisopliae, Beauveria bassiana and Paecilomyces lilacinus, respectively, and the number of deaths of infected termites in the above treatment and control groups was counted every day for 10 days. The results showed that the mortality rate of infected termites in the three treatment groups was significantly higher than that of the control group. It can be seen that SODsiRNA significantly improved the control effect of three biocontrol bacteria on termites.

[0023] The present invention has the following four obvious advantages: 1. Obtaining black-chested Reticulitermes SOD DNA sequence of genes, design SOD siRNA can specifically target target termites and closely related termites, reducing the impact on non-target organisms; 2. Compared with dsRNA in traditional RNAi methods, siRNA has a higher interference efficiency and a higher gene silencing effect; 3. SOD siRNA is a double-stranded RNA with a simple preparation method, strong specificity, and environmental friendliness. It can weaken the antioxidant capacity of termites through a simple feeding operation, and has an ideal effect of killing termites in synergistic with biocontrol bacteria, and has good research and application prospects. 4. SOD siRNA can be used in combination with a variety of biocontrol bacteria, such as Metarhizium, Beauveria bassiana and Paecilomyces oxysporum, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : SOD dsRNA and SOD siRNA, GFP dsRNA and GFP Gel electrophoresis of siRNA. Band 1 is GFP dsRNA; Band 2 is GFP siRNA; Band 3 is SOD dsRNA; Band 4 is SOD siRNA.

[0025] Figure 2 : Termites took 20 μg SOD dsRNA and 20 μg SOD siRNA 1 day later SOD Changes in gene expression. SOD dsRNA or SOD 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, SOD dsRNA in termites SOD There was no significant change in gene expression, but the same amount of food was consumed SOD siRNA in termites SOD Gene expression was significantly reduced; the bar graph is the mean ± standard error, * indicates P <0.05; ns indicates no significant difference.

[0026] Figure 3 :Termite feeding 20 μgSOD siRNA 1 day later SOD Changes in enzyme activity. SOD siRNA-treated termites were fed GFP siRNA termites were the control group; as shown in the figure, compared with the control group, SOD siRNA in termites SOD The enzyme activity was significantly reduced; the bar graph is the mean ± standard error, ** indicates P <0.01.

[0027] Figure 4 :3 kinds of biocontrol fungi culture and their ant-killing effect pictures. A and B represent the culture of Metarhizium anisopliae and its ant-killing effect pictures; C and D represent the culture of Beauveria bassiana and its ant-killing effect pictures; E and F represent the culture of Paecilomyces lilacinus and its ant-killing effect pictures.

[0028] Figure 5 : SOD The synergistic effect of siRNA on the killing of ants by Metarhizium anisopliae. SOD siRNA-treated termites were fed GFP siRNA termites were the control group; ** indicates P <0.01.

[0029] Figure 6 : SOD The synergistic effect of siRNA on the killing effect of Beauveria bassiana on ants. SOD siRNA-treated termites were fed GFP siRNA termites were the control group; ** indicates P <0.01.

[0030] Figure 7 : SOD The synergistic effect of siRNA on the killing effect of Paecilomyces lilacinus. SOD siRNA-treated termites were fed GFP siRNA termites were the control group; ** indicates P <0.01. DETAILED DESCRIPTION

[0031] Embodiment 1: SOD siRNA preparation process

[0032] 1. Extract total RNA from Reticulitermes nigrotermes and obtain termite cDNA library by reverse transcription.

[0033] 2. Using termite cDNA as template, SODGene-specific primers SEQ ID NO:3 (upstream primer: 5'-AACATT GCC CGC GCA TAA CC-3') and SEQ ID NO:4 (downstream primer: 5'-TCA TCT TCA ATG CGG GTCAG-3') were used to obtain the gene by PCR amplification. SOD The gene fragments were then separated and purified by agarose gel electrophoresis. SOD The gene fragment has a DNA sequence of SEQ ID NO: 2.

[0034] 3. Through TA cloning SOD The gene fragment was inserted into the PMD-18T plasmid, and then the target gene SOD 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.

[0035] 4. Take the competent cell solution after the above transformation, spread it evenly in the ampicillin resistance medium, and culture it at 37℃ for 24h.

[0036] 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. SOD The bacterial solution of the fragment is subjected to PCR and expanded culture.

[0037] 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. SOD The plasmid containing the target gene was obtained and stored at -20℃. SOD Purified plasmid fragment.

[0038] 7. Using the above plasmid as template, use the plasmid containing "transcription enhancer" and "T7 promoter" SOD The fragment-specific primers SEQ ID NO:5 (upstream primer: 5′-GAT CAC TAA TAC GAC TCA CTA TAG GGA ACA TTG CCC GCGCAT AAC C-3′) and SEQ ID NO:6 (downstream primer: 5′-GAT CAC TAA TAC GAC TCA CTA TAG GGTCAT GCA CCA CAA AAG CAC G-3′) were amplified by PCR. SOD dsRNA template. Collection SODdsRNA 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 SOD dsRNA template.

[0039] 8. Utilize the above SOD Obtaining dsRNA template and T7 in vitro transcription system SOD dsRNA. Collect the reaction system and add enzyme-free water to 300 μL. Then add 30 μL of sodium acetate, 300 μL of water-saturated phenol and 60 μL of chloroform / isoamyl alcohol (volume ratio 50:1) 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 minutes, and discard the supernatant. Finally, wash the precipitate with 75% alcohol, dry, and dissolve the precipitate to obtain SOD dsRNA.

[0040] 9. Digestion with RNase 3 SOD dsRNA SOD siRNA( Figure 1 ).

[0041] Example 2: Detection SOD Reticulitermes chinensis SOD Genetic influence

[0042] 20 μg of the above SOD dsRNA or SOD 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 GFP 1.5 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. SOD 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, SOD dsRNA treated termites SOD There was no significant change in gene expression ( Figure 2A, p = 0.249), but SOD siRNA treated termites SOD Gene expression was significantly reduced by 41.4% ( Figure 2 B, P<0.05), indicating SOD siRNA can better inhibit the SOD Gene expression.

[0043] Example 3: Detection SOD Effects of siRNA on the Antioxidant Activity of SOD in Reticulitermes nigroteres

[0044] 20 μg of the above SOD 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 feed on for 1 day. GFP siRNA was used to wet a 1.5 cm diameter circular filter paper and placed in a 3.5 cm diameter petri dish for feeding 9 black-chested termites for 1 day as a control group. Termite tissue fluid was extracted and stored in a -20°C refrigerator for later use. The SOD enzyme activity in termites of different treatment groups was detected using a SOD 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 SOD enzyme activity in the treated termites was significantly reduced by 38.1% (P<0.01), indicating that SOD siRNA can inhibit the ability of infected termites to resist oxidative damage.

[0045] Example 4: Detection SOD Effect of siRNA on the ant-killing effect of Metarhizium anisopliae

[0046] Cultivation of Metarhizium anisopliae (Met. anisopliae) using potato dextrose medium (PDA) Figure 4 A), after culturing for 2 weeks, collect the spores of Metarhizium anisopliae with 1% Tween 80 solution, prepare spore suspension, and store in a refrigerator at 4℃ for later use. 6 spores / mL.

[0047] 20 μg of the above SOD 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. GFPsiRNA 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 B. Figure 5 Compared with the control group infected termites [LT 50 : 7.936 (7.241-8.873)], the mortality rate of termites infected with fungi in the treatment group [LT 50 : 4.318 (2.951-5.369)] increased significantly (P<0.01), indicating SOD siRNA can significantly improve the effectiveness of Metarhizium anisopliae in killing termites.

[0048] Example 5: Detection SOD Effect of siRNA on the ant-killing effect of Beauveria bassiana

[0049] The method and steps are the same as those in Example 4, except that Metarhizium anisopliae is replaced by Beauveria bassiana ( Figure 4 C), then the result is Figure 4 D. Figure 6 As shown, the mortality rate of termites infected with fungi in the control group [LT 50 : 4.828 (4.192-5.346)], compared with the control group, the mortality rate of termites infected with fungi [LT 50 : 2.177 (1.928-2.418)] increased significantly (P<0.01), indicating SOD siRNA can significantly improve the effectiveness of Beauveria bassiana in killing termites.

[0050] Example 6: Detection SOD Effect of siRNA on the ant-killing effect of Paecilomyces lilacinus

[0051] The method and steps are the same as those in Example 4, except that Metarhizium anisopliae is replaced by Paecilomyces lilacinus ( Figure 4 E), then the result is Figure 4 E. Figure 7 As shown, the mortality rate of termites infected with fungi in the control group [LT 50 : 6.575 (5.274-8.784)], compared with the control group, the mortality rate of termites infected with fungi [LT 50 : 4.103 (3.153-5.262)] increased significantly (P<0.01), indicating SOD siRNA can significantly improve the effectiveness of Pseudomonas lilacinus in killing termites.

[0052] In summary,SOD siRNA has a wide range of synergistic effects: it has a high synergistic effect on Metarhizium, Beauveria bassiana and Paecilomyces. SOD siRNA+Metarhizium anisopliae” ([LT 50 : 4.318 (2.951-5.369)]), " SOD siRNA+Paecilomyces spp. (LT 50 : 4.103 (3.153-5.262)]) compared to the combination of SOD siRNA+Beauveria bassiana” ([LT 50 : 2.177(1.928-2.418)]) combination has the shortest half-lethal time and the highest ant killing efficiency.

Claims

1. SOD The gene, namely the superoxide dismutase gene, is characterized in that The DNA sequence of the gene is SEQ ID NO:

1.

2. A plasmid comprising SOD A plasmid containing a gene fragment, characterized in that The DNA sequence is SEQ ID NO:

4.

3. A SOD The dsRNA template is characterized in that SOD The DNA sequence of the dsRNA template is SEQ ID NO:

7.

4. A double-stranded nucleic acid SOD dsRNA, characterized in that Double-stranded nucleic acid SOD dsRNA, the RNA sequence is SEQ ID NO:

8.

5. A double-stranded nucleic acid SOD siRNA, characterized in that Using the double-stranded nucleic acid according to claim 4 SOD dsRNA was obtained by RNase III digestion SOD siRNA.

6. A double-stranded nucleic acid comprising the double-stranded nucleic acid according to claim 5 SOD siRNA products for controlling black-breasted termites.

7. A double-stranded nucleic acid comprising the double-stranded nucleic acid according to claim 6 SOD A product for controlling black-chested termites with siRNA as the active ingredient.

8. The product according to claim 7, characterized in that Double-stranded nucleic acid SOD The product for controlling black-breasted Reticulitermes is prepared by siRNA in cooperation with biocontrol fungi, wherein the biocontrol fungi include one or more of Metarhizium anisopliae, Beauveria bassiana, or Paecilomyces oxytoca.

9. A method for controlling black-chested termites, comprising the double-stranded nucleic acid according to any one of claims 6 to 8 SOD siRNA, characterized in that Here are the steps: (1) Double-stranded nucleic acid SOD siRNA is mixed with termite food and placed in a termite colony for termites to eat. SOD siRNA termites; (2) using a 0.1-2% Tween 80 solution to collect biocontrol fungus spores and prepare a biocontrol fungus spore suspension; (3) Using a suspension of biocontrol fungi spores to infect and feed on insects SOD siRNA for termites, thereby preventing and controlling termites.

10. The method for controlling Reticulitermes punctatus according to claim 9, characterized in that: Termite food includes cellulose-containing materials such as paper, wood chips, sawdust, or wood powder. SOD The siRNA is mixed with termite food by a mixing method of infiltration, smearing, or injection, and the biocontrol bacteria include one or more of Metarhizium anisopliae, Beauveria bassiana, or Paecilomyces oxysporum.

Images