Method for preventing and controlling aedes albopictus

Abstract

The invention discloses an aedes albopictus control method, which uses cationic polyacrylamide to prepare a carrier-dsRNA compound for treatment, and can effectively kill aedes albopictus in various insect states. The dsRNA used in the method targets the aedes albopictus V-ATP enzyme H subunit gene, and is safe to other non-target organisms; meanwhile, the adopted cationic polyacrylamide has the advantages of high nucleic acid loading efficiency and good anti-degradation performance, also has the characteristics of low toxicity, high efficiency, safety and environmental protection, is good in biocompatibility, and ensures the safety of a drug applicator in the drug application process. The action mechanism of the method is different from that of conventional pesticides, drug resistance is not prone to being generated, raw material resources are rich, cost is low, the preparation method is simple, and the method is suitable for large-scale application and popularization.

Description

Technical Field

[0001] The present invention relates to biological pesticides, in particular to a method for controlling Aedes albopictus. Background Art

[0002] The Aedes albopictus mosquito (Aedes albopictus) is a typical container-breeding mosquito, whose larvae often gather in small pools of water. They are highly adaptable and reproduce rapidly. As a key vector of pathogens such as dengue fever and Zika virus, their prevention and control are crucial for public health. While current mainstream chemical control methods, such as pyrethroids, can quickly kill mosquito larvae, their long-term use has led to a significant increase in insecticide resistance, necessitating the development of new, efficient, and sustainable control strategies.

[0003] RNA interference (RNAi) is a technology that specifically silences target genes through double-stranded RNA (dsRNA), and has been widely used in the field of pest control in recent years. The principle is that after dsRNA enters the insect body, it is cut into small interfering RNA (siRNA), which binds to the target gene mRNA and induces degradation, thereby blocking gene expression. This technology is highly specific and can achieve a high lethality rate by precisely selecting target genes (such as key genes for larval development or genes related to drug resistance), and it is relatively safe for humans and the environment. However, dsRNA is easily degraded by nucleases in the natural environment and the insect intestine, resulting in unstable RNAi efficiency. Therefore, the optimization of the delivery system has become a core challenge for the implementation of the technology.

[0004] Nanocarrier delivery systems combine negatively charged dsRNA with cationic polymers through electrostatic interactions, forming a stable "carrier-dsRNA complex" that protects dsRNA from enzymatic degradation while enhancing its ability to penetrate the larval cuticle or intestinal barrier, significantly improving RNAi efficiency. Commonly used cationic polymer carriers include the dendritic polymer PAMAM and the natural polysaccharide chitosan. PAMAM has a highly branched three-dimensional structure and controllable surface charge, allowing it to efficiently load dsRNA. However, its high synthesis cost and poor biodegradability may lead to environmental residue issues. Although chitosan is widely available and has good biocompatibility, its loading efficiency is low and it tends to aggregate into large particles in acidic environments, impairing its ability to penetrate the larval cuticle. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a low-cost, environmentally friendly method for controlling Aedes albopictus based on genetic engineering technology.

[0006] Technical solution: The method for controlling Aedes albopictus according to the present invention comprises the following steps:

[0007] (1) preparing a cationic polyacrylamide solution;

[0008] (2) adding a nucleic acid gene inhibitor to a cationic polyacrylamide solution to obtain a mixed preparation;

[0009] (3) Using the mixed preparation obtained in step 2 to treat Aedes albopictus.

[0010] Preferably, the molecular weight of the cationic polyacrylamide in step 1 is 500-600 kDa; the concentration of the cationic polyacrylamide solution is 0.01-0.03% (w / v), and the solvent is sodium acetate buffer.

[0011] Preferably, the nucleic acid gene inhibitor in step 2 is any one of dsRNA, siRNA, and shRNA, which inhibits the expression of V-ATPase H subunit and is dissolved in a sodium sulfate solution.

[0012] Preferably, the mass ratio of cationic polyacrylamide to nucleic acid gene inhibitor in the cationic polyacrylamide solution in step 2 is 0.25-1:1.

[0013] Preferably, after adding the nucleic acid gene inhibitor to the cationic polyacrylamide solution in step 2, the solution is heated and incubated for 30-90 seconds, and then vortexed at 2000-4000 rpm for 20-40 seconds; the heating and incubation temperature is 50-60°C.

[0014] Preferably, when the mixed preparation in step 3 is used to treat Aedes albopictus, the final concentration of the nucleic acid gene inhibitor is 120-600 μg / mL.

[0015] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: 1. It targets the H subunit gene of the V-ATPase of Aedes albopictus, is safe to other non-target organisms, has a significant killing effect on all stages of Aedes albopictus, and can be effectively used for the prevention and control of Aedes albopictus; 2. It uses cationic polyacrylamide to deliver dsRNA, which has higher loading efficiency than the common chitosan carrier and can effectively prevent dsRNA degradation; 3. It has the characteristics of low toxicity, high efficiency, safety and environmental protection, is safe to mammals such as humans and livestock, natural enemies of pests and other beneficial organisms, and has good biocompatibility; 4. The cationic polyacrylamide used has strong water solubility, greatly reduces damage to the environment, and ensures the safety of applicators during the application process; 5. The mechanism of action is different from that of conventional pesticides, and it is not easy to develop resistance to them; 6. The raw material resources are abundant, the cost is low, the preparation method is simple, and it is suitable for large-scale promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Figures 2 and 3 show the agarose gel electrophoresis results of dsRNA contained in the supernatant after the preparation of complexes with different vector-nucleic acid mass ratios, where A shows the result of cationic polyacrylamide-dsRNA complex and B shows the result of chitosan-dsRNA complex;

[0017] Figure 2 Figure 1 shows the agarose gel electrophoresis results of dsRNA contained in cationic polyacrylamide-dsRNA complexes with a carrier-to-nucleic acid mass ratio of 1:1 and chitosan-dsRNA complexes with a carrier-to-nucleic acid mass ratio of 2:1 after storage at room temperature. Lanes 1 and 4 represent independent dsRNA without a carrier, lanes 2 and 5 represent chitosan-dsRNA, and lanes 3 and 6 represent cationic polyacrylamide-dsRNA.

[0018] Figure 3 Fluorescence images of the midgut of Aedes albopictus larvae transfected with cationic polyacrylamide-dsRNA complexes and chitosan-dsRNA complexes labeled with nucleic acid dyes;

[0019] Figure 4 Schematic diagram of the principle of using cationic polyacrylamide loaded with dsRNA to kill Aedes albopictus larvae;

[0020] Figure 5 This is a statistical graph showing the killing of adult Aedes albopictus mosquitoes by cationic polyacrylamide-loaded dsRNA. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below.

[0022] Example 1 Preparation and Characterization of Cationic Polyacrylamide (CPAM) Loaded dsRNA

[0023] 1. Preparation of dsRNA

[0024] (1) A gene fragment of the VATP-h subunit of Aedes albopictus V-ATPase (reference sequence: NCBI Reference Sequence: XM_029864458.2) was amplified and ligated into the pET-2p dsRNA expression vector plasmid, and then transformed into DH5a competent cells;

[0025] (2) The transformed bacterial solution was inoculated onto a plate containing 100 μg / mL kanamycin, and the positive transformants that grew were selected for sequencing verification;

[0026] (3) Amplify and sequence the positive transformants and use Plasmids were extracted using Plasmid DNA Mini Kit 1 (Omega);

[0027] (4) The extracted plasmid was transformed into HT115(DE3) competent cells, and the transformed bacterial solution was inoculated onto a plate containing 100 μg / mL kanamycin. The positive HT115(DE3) transformants that grew were selected for sequencing verification;

[0028] (5) Amplify the HT115(DE3)-pET-2p dsRNA that has been successfully verified by sequencing. When the bacterial solution OD 600 =0.9, add 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and continue to culture at 37°C with shaking at 200 rpm for 4 h;

[0029] (6) Take 1 mL of IPTG-induced bacterial culture and centrifuge at 10,000 g for 5 min at 4°C. Discard the supernatant and add 70 μL of STE buffer (Solarbio) to resuspend the pellet. Then add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) (Solarbio) and shake vigorously for 150 s. Then centrifuge at 13,000 g for 5 min at 4°C. Aspirate the top layer of liquid and collect it in a sterile, enzyme-free PCR tube to obtain the purified dsRNA.

[0030] 2. Preparation of cationic polyacrylamide loaded dsRNA

[0031] (1) Dissolving cationic polyacrylamide in sodium acetate buffer at pH 4.5 to obtain a working solution with a concentration of 0.02% (w / v);

[0032] (2) 20, 40, 60, and 80 μg of dsRNA were dissolved in 100 μL of 50 mM Na2SO4, respectively, and added to 100 μL of the aforementioned cationic polyacrylamide working solution. After incubation at 55°C for 60 s, the mixture was quickly vortexed at 3000 rpm for 30 s to obtain complexes with different cationic polyacrylamide-dsRNA mass ratios (dsRNA / CPAM).

[0033] 3. Characterization of cationic polyacrylamide loaded dsRNA

[0034] 10, 20, 40, and 60 μg of dsRNA were dissolved in 100 μL of 50 mM Na2SO4 and added to 100 μL of 0.02% (w / v) chitosan (molecular weight 50-150 kDa) sodium acetate buffer (pH = 4.5). After incubation at 55°C for 60 seconds, the mixture was vortexed at 3000 rpm for 30 seconds to obtain complexes with different chitosan-dsRNA mass ratios (dsRNA / Chitosan).

[0035] The cationic polyacrylamide-dsRNA complexes and chitosan-dsRNA complexes obtained above were centrifuged at 13,000 rpm for 15 min, and the supernatants were collected and loaded onto 1% agarose gel. The dsRNA in the supernatants was detected by electrophoresis.

[0036] The results are as follows Figure 1 As shown, no obvious bands were observed for cationic polyacrylamide-dsRNA at a mass ratio of 1:1, indicating complete loading of dsRNA; while chitosan-dsRNA still had obvious bands at a mass ratio of 1:1, but no obvious bands were observed at a mass ratio of 2:1, indicating that chitosan and nucleic acid were completely loaded at a mass ratio of 2:1, proving that cationic polyacrylamide has higher loading efficiency.

[0037] According to the above steps, a cationic polyacrylamide-dsRNA complex with a mass ratio of 1:1 and a chitosan-dsRNA complex with a mass ratio of 2:1 were prepared to achieve a final dsRNA concentration of 200 mg / L. Naked dsRNA alone (naked dsRNA) was also prepared as a control. The samples were stored at room temperature for 7 or 14 days. The samples were loaded onto a 1% agarose gel and the dsRNA was detected by electrophoresis.

[0038] The results are as follows Figure 2 As shown, the uncarrier dsRNA alone had undergone significant degradation at room temperature on the 7th day, and the fluorescence signal was weak, while the dsRNA loaded with cationic polyacrylamide still maintained a similar fluorescence intensity on the 14th day, and compared with chitosan-dsRNA, the fluorescence signal did not decrease significantly, that is, cationic polyacrylamide has a better dsRNA protection effect, preventing dsRNA from degradation at room temperature.

[0039] Example 2 Nucleic acid stain-labeled dsRNA to observe transfection ability in the midgut of Aedes albopictus larvae

[0040] (1) Using 4S Red Plus nucleic acid stain to label the dsRNA obtained in Example 1;

[0041] (2) Referring to the steps of Example 1, the vector and dsRNA were mixed under conditions where the mass ratio of cationic polyacrylamide to dsRNA was 1:1 and the mass ratio of chitosan to dsRNA was 2:1. After incubation at 55°C for 60 seconds, the mixture was rapidly vortexed at 3000 rpm for 30 seconds to obtain different vector-dsRNA complexes;

[0042] (3) After immersing the third-instar larvae of Aedes albopictus for 24 hours, the larvae were taken out and washed three times with 70% ethanol and double-distilled water to remove the vector-dsRNA complex attached to the surface of the larvae. The fluorescence in the larvae was observed using a fluorescence microscope.

[0043] The results are as follows Figure 3 As shown in the figure, the fluorescence signal of dsRNA without a carrier is weak in the larval intestine, indicating that the dsRNA has been degraded and cannot be effectively transfected; while the dsRNA loaded with chitosan and cationic polyacrylamide still has a high fluorescence intensity, indicating that cationic polyacrylamide has a similar ability to chitosan to protect dsRNA from being degraded by larval intestinal enzymes, which can effectively improve the transfection efficiency and enhance the silencing efficiency of dsRNA.

[0044] Example 3 Evaluation of the effect of cationic polyacrylamide loaded with dsRNA on the treatment of Aedes albopictus larvae

[0045] The bioactivity against third instar larvae of Aedes albopictus was determined by the larval immersion method.

[0046] The dsRNA was set to five concentration gradients: 120, 240, 360, 480, and 600 μg / mL. The carrier was fully loaded, i.e., the mass ratio of cationic polyacrylamide to dsRNA was 1:1, and the mass ratio of chitosan to dsRNA was 2:1. Unloaded dsRNA was used as a blank control. Third-instar larvae of Aedes albopictus were treated with the above dilutions in a greenhouse at 25±1°C and a relative humidity of 60-80%. Mortality was examined after 24 hours. Each treatment was repeated five times, with 20 larvae per treatment. LC values ​​were calculated using SPSS software (v22.0). 50 The toxicity regression curve is shown in Table 1 below.

[0047] Table 1 LC of different carriers 50 , 95% confidence interval and toxicity regression curve

[0048] Pharmacy <![CDATA[LC 50 ]]> 95% confidence interval Virulence regression curve <![CDATA[R 2 ]]> dsVATP-h 394 μg / mL 306-505 μg / mL Y=3.1X-8.1 0.929 Chitosan-dsVATP-h complex 316 μg / mL 248-382 μg / mL Y=4.8X-5.2 0.944 CPAM-dsVATP-h complex 259 μg / mL 233-284 μg / mL Y=4.3X-10.3 0.911

[0049] In the toxicity regression curve, Y is the probability value of mortality and X is the logarithm of the administered dose.

[0050] As can be seen from the table above, compared with using dsRNA alone to treat larvae, cationic polyacrylamide has a significant synergistic effect, and compared with the common carrier chitosan, the synergistic effect is even more obvious. It can effectively enhance the toxicity of dsRNA to Aedes albopictus larvae. The process diagram of cationic polyacrylamide loaded with dsRNA to kill Aedes albopictus larvae is shown below. Figure 4 shown.

[0051] Example 4 Evaluation of the Effect of Cationic Polyacrylamide-Loaded dsRNA on the Treatment of Aedes albopictus Adults

[0052] According to the method described in Example 1, cationic polyacrylamide-loaded dsRNA (dsVATPase-h) or cationic polyacrylamide-loaded control dsRNA (dsGFP) with a vector-dsRNA mass ratio of 1:1 was prepared.

[0053] Two-day-old, unmated Aedes albopictus mosquitoes were anesthetized by freezing in a -20°C refrigerator and placed in a lateral position on a pre-cooled 0°C metal bath. Microinjection was performed into the scale crevice in the middle of the left thorax, with the needle held perpendicular to the side of the mosquito. Thirty adult mosquitoes were injected with either cationic polyacrylamide-dsVATPase-h or cationic polyacrylamide-dsGFP, at a dose of 200 ng per mosquito. Mortality was recorded every 24 hours for seven consecutive days following injection.

[0054] The results are as follows Figure 5 As shown, the cationic polyacrylamide-dsVATPase-h treatment group had a high mortality rate, indicating that dsVATP-h has a good lethal effect on adult mosquitoes and is an excellent target for controlling Aedes albopictus. Combined with the results of Example 3, it can be seen that the cationic polyacrylamide-dsRNA complex has a significant killing effect on all stages of Aedes albopictus.

Claims

1. A method for controlling Aedes albopictus, characterized in that the steps include: (1) preparing a cationic polyacrylamide solution; (2) adding a nucleic acid gene inhibitor to a cationic polyacrylamide solution to obtain a mixed preparation; (3) Using the mixed preparation obtained in step 2 to treat Aedes albopictus.

2. The method for controlling Aedes albopictus according to claim 1, characterized in that: The molecular weight of the cationic polyacrylamide in step 1 is 500-600 kDa.

3. The method for controlling Aedes albopictus according to claim 1, characterized in that: The concentration of the cationic polyacrylamide solution in step 1 is 0.01-0.03% (w / v), and the solvent is sodium acetate buffer.

4. The method for controlling Aedes albopictus according to claim 1, characterized in that: The nucleic acid gene inhibitor in step 2 is any one of dsRNA, siRNA, and shRNA.

5. The method for controlling Aedes albopictus according to claim 4, characterized in that: The nucleic acid gene inhibitor inhibits the expression of V-ATPase H subunit.

6. The method for controlling Aedes albopictus according to claim 1, characterized in that: The mass ratio of cationic polyacrylamide to nucleic acid gene inhibitor in the cationic polyacrylamide solution in step 2 is 0.25-1:

1.

7. The method for controlling Aedes albopictus according to claim 1, characterized in that: The nucleic acid gene inhibitor in step 2 is dissolved in a sodium sulfate solution.

8. The method for controlling Aedes albopictus according to claim 1, characterized in that: After adding the nucleic acid gene inhibitor to the cationic polyacrylamide solution in step 2, the solution was heated and incubated for 30-90 seconds, and then vortexed at 2000-4000 rpm for 20-40 seconds.

9. The method for controlling Aedes albopictus according to claim 8, characterized in that: The temperature of the heating incubation is 50-60°C.

10. The method for controlling Aedes albopictus according to claim 1, characterized in that: When the mixed preparation in step 3 is used to treat Aedes albopictus, the final concentration of the nucleic acid gene inhibitor is 120-600 μg / mL.

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