DsDNA, CS-TPP-dsDNA and preparation method and application thereof

Abstract

The invention belongs to the technical field of insect prevention and control, and particularly relates to dsDNA and CS-TPP-dsDNA as well as a preparation method and application thereof. The dsDNA disclosed by the invention is obtained by annealing oligonucleotide as shown in SEQ ID NO.1 and oligonucleotide as shown in SEQ ID NO.2; after the HaCYP6AE19 is interfered by adopting the dsDNA, the sensitivity of the cotton bollworm to the lambda-cyhalothrin can be improved, and the use of chemical agents is reduced. However, the intestinal fluid and lymph of the cotton bollworm can degrade the dsDNA, the CS-TPP-dsDNA prepared by wrapping the dsDNA with the chitosan nano-carrier can effectively solve the problem that the dsDNA is easy to degrade, the interference efficiency of feeding is improved, and a new thought is provided for prevention and treatment of the cotton bollworm.

Description

Technical Field

[0001] The present invention belongs to the technical field of insect control, and in particular relates to dsDNA, CS-TPP-dsDNA and a preparation method and application thereof. Background Art

[0002] Gene interference technology, through the use of specific double-stranded RNA (dsRNA) sequence regions, can highly specifically degrade the mRNA of target genes, thereby precisely silencing specific genes in pests. This means that it only works on specific genes in target pests and has little effect on non-target organisms (such as beneficial insects, other animals, and humans). RNAi technology has been successfully applied to control a variety of important agricultural pests, but dsRNA interference has been less effective against Lepidoptera insects. Therefore, the search for new, highly effective, and safe, green pesticides is a key development direction for the pesticide industry. Summary of the Invention

[0003] During the research on the prevention and control of cotton bollworm, the inventors found that inducing the expression of HaCYP6AE19 in cotton bollworm can enhance resistance to cyhalothrin. Therefore, they tried to silence the HaCYP6AE19 gene using gene interference technology, but found that dsRNA had the problem of low silencing efficiency.

[0004] In order to promote the application of gene interference technology in the prevention and control of cotton bollworm, the present invention provides a dsDNA, CS-TPP-dsDNA and a preparation method and application thereof.

[0005] In a first aspect, the present invention provides a dsDNA for silencing the HaCYP6AE19 gene of cotton bollworm, which is obtained by annealing the oligonucleotide shown in SEQ ID NO.1 and the oligonucleotide shown in SEQ ID NO.2.

[0006] This method utilizes the complex detoxification enzyme system evolved over time in insects, using long-term stress treatment with a diet containing lambda-cyhalothrin to screen for detoxification enzyme genes that metabolize it. This method demonstrates robust logic and high feasibility. The dsDNA described in this method has excellent silencing efficiency and effective rate for the HaCYP6AE19 gene, laying the foundation for the development of a new, green pesticide for controlling cotton bollworms.

[0007] In a second aspect, the present invention provides a CS-TPP-dsDNA for controlling insects, which is obtained by encapsulating the dsDNA in a chitosan nanocarrier.

[0008] In a third aspect, the present invention provides a method for preparing the CS-TPP-dsDNA, characterized in that it comprises the following steps: Chitosan and sodium tripolyphosphate are used as raw materials, and ionic cross-linking occurs in a solvent system through electrostatic attraction to obtain a chitosan nanocarrier suspension; The dsDNA is added to the chitosan nanocarrier suspension, incubated and then mixed evenly to obtain the CS-TPP-dsDNA.

[0009] In some specific embodiments, the mass ratio of chitosan to sodium tripolyphosphate is 5-7:1.

[0010] In some embodiments, the incubation condition is 54.5-55.5° C. for 29-30 min.

[0011] In a third aspect, the present invention provides use of the dsDNA or the CS-TPP-dsDNA in preparing an insect control agent.

[0012] In some embodiments, the insect is cotton bollworm.

[0013] In a fourth aspect, the present invention provides the use of the CS-TPP-dsDNA in improving the sensitivity of cotton bollworm to lambda-cyhalothrin.

[0014] The present invention has the following beneficial effects: The dsDNA of the present invention has a good interference effect on the cotton bollworm HaCYP6AE19. The CS-TPP-dsDNA of the present invention uses chitosan as a raw material, is inexpensive, has a relatively simple synthesis method, and exhibits low toxicity, good biocompatibility, and excellent biodegradability. The CS-TPP-dsDNA of the present invention has a better interference effect on HaCYP6AE19 than naked dsDNA and siRNA. Indoor bioassay results show that feeding CS-TPP-dsDNA can enhance the sensitivity of test insects to lambda-cyhalothrin. Its application in the field can reduce the dosage of lambda-cyhalothrin. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the result of agarose gel electrophoresis detection of dsDNA.

[0016] Figure 2 This is the detection of the silencing efficiency of HaCYP6AE19 in the test insects 2 days after dsDNA injection.

[0017] Figure 3 Agarose gel electrophoresis was used to detect the formation of CS-TPP-dsDNA at different ratios.

[0018] Figure 4The relative expression levels of HaCYP6AE19 after feeding with naked dsDNA and CS-TPP-dsDNA complex for 48 h (A), and the relative expression levels of HaCYP6AE19 after feeding with naked siRNA for 48 h (B). DETAILED DESCRIPTION

[0019] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0020] Abbreviations and their full names in the context of this invention: CS: Chitosan.

[0021] TPP: sodium tripolyphosphate.

[0022] The oligonucleotide sequences involved in the following embodiments of the present invention are shown in Table 1: Table 1 Primer sequences Example 1: Synthesis of dsDNA interfering with the HaCYP6AE19 gene and determination of silencing efficiency.

[0023] 1. Experimental methods (1) Synthesis of dsDNA The following system was prepared to generate dsDNA: 10 μL of dsDNA-F, 10 μL of dsDNA-R, 50 μL of 2× Oligo annealing buffer, and 30 μL of enzyme-free water. The oligonucleotide mixture was heated at 95°C for 8 min and then slowly cooled to room temperature (at a rate of 0.1°C / s to 25°C). The concentration was verified and determined by agarose gel electrophoresis.

[0024] (2) Determination of silencing efficiency Third-instar susceptible cotton bollworms of roughly identical growth were placed on ice for anesthesia. Forceps were used to gently grasp the tail of the bollworm. A needle was used to gently pierce the epidermis at the first abdominal foot and slowly inject dsDNA (270 ng / μL). A total of 12 test insects were injected, and three replicates were used as controls, with six test insects per replicate. 48 hours after dsDNA injection, the silencing efficiency and effectiveness of the HaCYP6AE19 gene were determined by RT-qPCR. A single test insect was considered acceptable for a silencing efficiency of at least 50% and an effectiveness of at least 70%.

[0025] Take multiple cotton bollworms, dissect and remove midgut fat bodies and mix them. Use RNAiso Plus to extract total RNA.Ⓡ The 1st Strand cDNA Synthesis SuperMix for qPCR Reverse transcribed the cDNA into cDNA. The RT-qPCR reaction system consisted of 20 μL: 10 μL Hieff qPCR SYBR Green Master Mix, 0.6 μL each of dsDNA-F and dsDNA-R, 1 μL template, and ddH2O to 20 μL. The amplification program was as follows: 1 cycle at 95°C for 10 min; 40 cycles at 95°C for 15 s and 60°C for 60 s. RPS15 and RPL32 were used as internal reference genes, and 2 -ΔΔCt The relative expression of genes was calculated using the method of . The experiment was repeated three times.

[0026] 2. Experimental results like Figure 1 As shown, the synthesized dsDNA was detected by agarose gel, and the result showed that the size of the dsDNA was 19 bp and the target band size was correct.

[0027] like Figure 2 As shown, RT-qPCR was used to examine the silencing efficiency and effectiveness of dsDNA against HaCYP6AE19. Two days after dsDNA injection, HaCYP6AE19 expression levels in the test insects were 0.04, 0.09, 0.01, 0.03, 0.67, 0.13, 0.02, 0.60, 0.31, 0.10, 0.01, and 0.84 times higher than those in the control group, respectively. This marked a significant decrease of 77% compared with the control group (p < 0.05). Silencing efficiency exceeded 50% in 9 of the 12 test insects, resulting in a silencing effectiveness rate of 75%. These results demonstrate that dsDNA has a good silencing efficiency and effectiveness for HaCYP6AE19.

[0028] Example 2: Synthesis and feeding method of CS-TPP-dsDNA Feeding cotton bollworms.

[0029] 1. Experimental methods (1) Synthesis of CS-TPP-dsDNA Medium molecular weight CS (75% deacetylated, Aladdin CAS: 9012-76-4) was dissolved in 1% acetic acid (w / v) to prepare a 10 mg / ml solution. TPP (Aladdin CAS: 7758-29-4) was dissolved in deionized water (1 mg / ml). Nanoparticle formation was observed by adding 10 mL of TPP solution to 1 mL, 3 mL, 5 mL, and 7 mL of CS solution under magnetic stirring at room temperature. After 2 hours of magnetic stirring, 1 mL of the nanosuspension was aspirated and centrifuged at 12,000 x g for 10 minutes. The pellet was then resuspended in 1 mL of deionized water and sonicated for 10 minutes using a bath sonicator at 35 kHz and 120 W of power. 200 μL of dsDNA (270 ng / μL) was added to each of the four solutions, incubated at 55°C for 30 minutes, and vortexed for 30 seconds. The nanoparticles were then detected by agarose gel electrophoresis.

[0030] (2) Synthetic siRNA siRNA-1 to siRNA-4 were used to synthesize siRNAs that targeted and inhibited HaCYP6AE19, and siGFP-1 to siGFP-4 were used to synthesize control siGFP.

[0031] Prepare the following system to generate sense or antisense RNA templates: 10 μL of sense strand, 10 μL of antisense strand, 50 μL of 2× Oligo Annealing Buffer, and 30 μL of enzyme-free water. Heat the oligonucleotide mixture at 93°C for 4 minutes, then slowly cool the mixture to room temperature. Then, use the T7 RiboMAX™ Express RNAi System kit for in vitro transcription and synthesis of siRNA. The specific steps are as follows:

[0032] Mix 10 μL of RiboMax™ Express T7 2× Buffer, 2 μL of annealed oligonucleotide template DNA, 2 μL of Enzyme Mix T7 Express, and 6 μL of nuclease-free water, then incubate at 37°C for 6 h. Add 1 μL of RQ1 RNase-Free DNase to each 20 μL transcription reaction and incubate at 37°C for 30 min. Combine the separate sense and antisense strand reactions and incubate at 70°C for 10 min, then slowly cool to room temperature (0.1°C / s to 25°C). Next, add 0.1 volume of 3 M sodium acetate (pH 5.2) and 1 volume of isopropanol, mix, and incubate on ice for 5 min. Transfer the entire mixture to a 1.5 mL enzyme-free centrifuge tube and centrifuge at maximum speed for 10 min. Carefully aspirate the supernatant, and wash the pellet with 0.5 mL of pre-chilled 70% ethanol. After washing, the supernatant was removed and the tube was air-dried for 2 min, then 100 μL of enzyme-free water was added to resuspend the RNA and stored at -80°C.

[0033] (3) Feeding method: Feeding cotton bollworms The feeding DNAi experiment used a susceptible third-instar strain of cotton bollworm. 6 μL each of synthetic dsDNA and CS-TPP-dsDNA was evenly applied to an artificial diet (5 mm × 5 mm × 5 mm in size, with 1 μL applied evenly on each side). Samples were collected 48 hours after feeding. The midgut fat bodies were dissected and pooled, and total RNA was extracted, reverse-transcribed into cDNA, and analyzed by RT-qPCR.

[0034] 2. Experimental results like Figure 3 As shown, the synthesized CS-TPP-dsDNA with different ratios was detected by agarose gel, and the brightness of the bands was all in the spotting wells, indicating that all dsDNA was loaded.

[0035] like Figure 4 As shown in Figure A, the relative expression of HaCYP6AE19 in the naked dsDNA-fed group was downregulated by 24%, and that in the CS-TPP-dsDNA-fed group was downregulated by 65%. Compared with the control group, the expression level of the HaCYP6AE19 gene in the CS-TPP-dsDNA-fed group was significantly decreased, while there was no significant difference between the naked dsDNA-fed group and the control group. Figure 4 Figure B shows that the relative expression of HaCYP6AE19 in the naked dsDNA-fed group was downregulated by 16%, with no significant difference compared to the control group. These results indicate that feeding CS-TPP-dsDNA complexes significantly improved the interference efficiency compared to the naked dsDNA and naked siRNA-fed groups.

[0036] Example 3: Indoor bioassay.

[0037] 1. Experimental methods The experiment was conducted on third-instar susceptible cotton bollworm strains. After two days of feeding with dsDNA, CS-TPP-dsDNA, and siRNA, the test insects were subjected to a leaf dip method for the bioassay of cyhalothrin. Green cabbage leaves were used as test leaves and punched into 0.8 cm round cakes using a hole punch. 0.7 g of cyhalothrin technical was weighed into a 50 mL volumetric flask and diluted with acetone to a stock solution with a concentration of 13,500 mg / L. The stock solution was then serially diluted with 0.1% Tween 80 to concentrations of 0, 1.67, 5, 15, 45, and 135 mg / L. Thirty test insects were used for each concentration, and the experiment was repeated three times. After 72 h, the mortality of the test insects was counted, and the adjusted mortality rate and the LC value of each treatment were calculated. 50 .

[0038] 2. Experimental results The changes in the sensitivity of the test insects to cyhalothrin after feeding with different treatment feeds are shown in Table 2. The LC 50 The LC of the test insects fed with siRNA feed was 35.55 mg / L. 50 The LC of the test insects fed with dsDNA feed was 30.43 mg / L. 50 The LC of the test insects fed with CS-TPP-dsDNA feed was 30.00 mg / L. 50 The LC of the control group was 13.52 mg / L. 50 The results showed that the sensitivity of the test insects to cyhalothrin was enhanced when they were fed the feed containing CS-TPP-dsDNA.

[0039] Table 2 Changes in sensitivity of test insects to lambda-cyhalothrin after feeding with different treatments It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0040] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. A method for silencing cotton bollworms HaCYP6AE19 The dsDNA of a gene is characterized in that It is obtained by annealing the oligonucleotide shown in SEQ ID NO.1 and the oligonucleotide shown in SEQ ID NO.

2.

2. A CS-TPP-dsDNA for controlling insects, characterized in that: The method is obtained by encapsulating the dsDNA according to claim 1 by chitosan nanocarriers.

3. The method for preparing CS-TPP-dsDNA according to claim 2, characterized in that: The following steps are involved: Chitosan and sodium tripolyphosphate are used as raw materials, and ionic cross-linking occurs in a solvent system through electrostatic attraction to obtain a chitosan nanocarrier suspension; The dsDNA according to claim 1 is added to the chitosan nanocarrier suspension, incubated and mixed evenly to obtain the CS-TPP-dsDNA.

4. The method for preparing CS-TPP-dsDNA according to claim 3, wherein: The mass ratio of chitosan to sodium tripolyphosphate is 5-7:

1.

5. The method for preparing CS-TPP-dsDNA according to claim 3, wherein: The incubation conditions are 54.5~55.5℃, 29~30min.

6. Use of the dsDNA according to claim 1 or the CS-TPP-dsDNA according to claim 2 in the preparation of an insect control agent.

7. The use according to claim 6, characterized in that The insect is cotton bollworm.

8. Use of the CS-TPP-dsDNA according to claim 2 in improving the sensitivity of cotton bollworm to lambda-cyhalothrin.

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