Double-stranded circular DNA and application thereof in biological control of cotton bollworm

Abstract

The invention discloses double-stranded circular DNA and application thereof in biological prevention and control of cotton bollworm, and relates to the technical field of genetic engineering and agricultural biological prevention and control, the two circular DNA (recombinant plasmids: pBE-cDNA4-lhAChE and pBE-cDNA4-dsAChE) stably generate double-stranded RNA and long hairpin RNA in an insect body and activate RNA interference (RNAi) effect by targeting a key gene of the cotton bollworm, so that the target gene is silenced; and the compound can also have synergistic interaction with viruses (HaNPV), so that the growth and development of cotton bollworms are inhibited. The invention has the advantages of high stability, strong delivery efficiency, environmental friendliness and the like, and is suitable for biological control of cotton bollworm. The technology can reduce the use of chemical pesticides and promote the sustainable development of agriculture.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and insect biological control technology, specifically involving a double-stranded circular DNA and its application in the biological control of cotton bollworm. Background Technology

[0002] cotton bollworm ( Helicoverpa armigera Chemical pests are serious pests of agricultural crops, especially cotton. While traditional chemical pesticides are effective, their negative environmental impacts and the development of pesticide resistance in pests necessitate a greater need for greener and more sustainable control methods.

[0003] RNA interference (RNAi) technology effectively inhibits the expression of target genes using specific double-stranded RNA (dsRNA) or long hairpin RNA (lhRNA) techniques, and is gradually becoming a green and environmentally friendly alternative in pest control.

[0004] In existing technologies, linear dsRNA has poor stability in insects and requires complex vector systems (such as nanoparticles or viral vectors), which are costly. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a double-stranded circular DNA.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a double-stranded circular DNA, wherein the double-stranded circular DNA comprises recombinant plasmid pBE-cDNA4-lhAChE and recombinant plasmid pBE-cDNA4-dsAChE; wherein, The nucleotide sequence of the recombinant plasmid pBE-cDNA4-lhAChE is shown in SEQ ID NO:1; The nucleotide sequence of the recombinant plasmid pBE-cDNA4-dsAChE is shown in SEQ ID NO:2.

[0009] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of double-stranded circular DNA in the biological control of cotton bollworm, wherein: it includes delivery to cotton bollworms via feeding or a combination of virus and feeding, inducing the silencing of target genes and inhibiting the growth and development of cotton bollworms.

[0010] Another object of the present invention is to overcome the shortcomings of the prior art and provide a composition for inhibiting the growth and development of cotton bollworm, comprising cotton bollworm nucleopolyhedrovirus HaNPV and the double-stranded circular DNA, both of which synergistically inhibit the growth and development of cotton bollworm.

[0011] Beneficial effects of this invention: (1) This invention provides two methods for preparing double-stranded circular DNAs that can efficiently express long hairpin RNA (lhRNA) and double-stranded RNA (dsRNA) in cotton bollworms and their applications. The two circular DNAs (recombinant plasmids: pBE-cDNA4-lhAChE and pBE-cDNA4-dsAChE) target key genes in cotton bollworms, stably producing double-stranded RNA and long hairpin RNA in the insect, activating RNA interference (RNAi) effect, leading to target gene silencing, and can also synergistically enhance with viruses (HaNPV), thereby inhibiting the growth and development of cotton bollworms.

[0012] (2) This invention has the advantages of high stability, strong delivery efficiency and environmental friendliness. It is suitable for biological control of cotton bollworm. This technology can reduce the use of chemical pesticides and promote sustainable agricultural development. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Wherein: Figure 1 This is a map of the recombinant plasmid vector pBE-cDNA4-lhAChE in an embodiment of the present invention.

[0014] Figure 2 This is a map of the recombinant plasmid vector pBE-cDNA4-dsAChE in an embodiment of the present invention.

[0015] Figure 3 This is a flowchart illustrating the construction process of two RNA interference plasmids in this embodiment of the invention.

[0016] Figure 4 This is a schematic diagram of the structures of two RNA interference plasmids in an embodiment of the present invention.

[0017] Figure 5 This invention compares the mortality rates of cotton bollworms of different ages when fed RNAi plasmids until pupation in this embodiment. Different letters (a, b, c) indicate significant differences between groups (P < 0.05).

[0018] Figure 6This is a heatmap showing the spatiotemporal distribution of the lhAChE plasmid in the body of cotton bollworm in an embodiment of the present invention. The figure shows the distribution changes of the lhAChE plasmid in different tissues (midgut, fat body, hemolymph and brain) over time (6h, 12h, 24h and 48h). The color intensity represents the plasmid DNA content (×10^4 copies / mg tissue), and the values ​​are the mean ± standard error.

[0019] Figure 7 This is a graph showing the effect of RNA interference plasmid and HaNPV treatment alone and in combination on the survival rate of cotton bollworm larvae in the embodiments of the present invention. In the graph, A: the effect of lhAChE and HaNPV combined treatment on the survival rate of cotton bollworm larvae; B: the effect of dsAChE and HaNPV combined treatment on the survival rate of cotton bollworm larvae. ** indicates P<0.01, and is a comparison with the theoretical superposition value.

[0020] Figure 8 This is a diagram showing typical pathological symptoms of larvae in the combined treatment group in this embodiment of the invention.

[0021] Figure 9 The figure shows the relative expression levels of the AChE gene in different treatment groups of cotton bollworm in the embodiments of the present invention (after 72 hours of treatment). A: The effect of combined treatment of lhAChE and HaNPV on AChE gene expression; B: The effect of combined treatment of dsAChE and HaNPV on AChE gene expression. **** indicates P<0.0001, compared with the corresponding RNAi treatment group alone. Detailed Implementation

[0022] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0023] Example 1 Construction and identification of PBE-cDNA4 plasmid vector: This embodiment aims to construct a basic expression vector pBE-cDNA4 containing a CMV promoter and necessary selection markers for subsequent cloning of RNA interference elements. This vector is constructed by fusing a specific fragment of PBE-sDNA4 and pcDNA4 / HisMax B, and then ligated using a double enzyme digestion strategy with MluI and ScaI to form the fusion plasmid pBE-cDNA4. Among them, plasmids PBE-sDNA4 and pcDNA4 / HisMax B were purchased from Baosai Plasmid and Straw Resource Company. The nucleotide sequence of PBE-sDNA4 is shown in SEQ ID NO:11, and the nucleotide sequence of pcDNA4 HisMax B is shown in SEQ ID NO:12.

[0024] 1.1. Enzyme digestion of PBE-sDNA4 vector and pcDNA4 / HisMax B vector The circular plasmids PBE-sDNA4 and pcDNA4 / HisMax B were double-digested to obtain the target fragment for ligation. The digestion reaction system is shown in Table 1.1.

[0025] Table 1.1 Enzyme digestion system of pcDNA4 / HisMax B vector

[0026] Prepare the reaction system, mix and centrifuge, then incubate at 37°C. After enzyme digestion, heat inactivate at 65°C for 10 min to terminate the reaction and avoid interference from subsequent ligation.

[0027] Take 3 μL of the enzyme digestion product for agarose gel electrophoresis identification. After electrophoresis, remove the gel and place it in an imaging device to observe and record the DNA bands.

[0028] 1.2 Enzyme digestion fragment purification and ligation The target restriction fragments (3118 bp and 4588 bp, respectively) were recovered and purified using an agarose gel DNA recovery kit. The nucleotide sequence of the 3118 bp fragment is shown in SEQ ID NO:13, and the nucleotide sequence of the 4588 bp fragment is shown in SEQ ID NO:14. The two purified target fragments were ligated using T4 DNA ligase. The ligation reaction system is shown in Table 1.2.

[0029] Table 1.2 Enzyme-linked reaction system

[0030] Configure the system according to the table above, mix it slightly, centrifuge, and incubate overnight at 16°C.

[0031] 1.3 Transformation of ligation products into Escherichia coli Transformation of ligation product: Add 10 μL of ligation product to 100 μL of thawed TOP10 competent cells, mix gently, and incubate on ice for 30 min. Heat shock at 42℃ for 90 s, then quickly transfer to ice and incubate for 2 min. Add 700 μL of antibiotic-free LB medium and incubate at 37℃ and 150 rpm for 60 min. Spread 150 μL of bacterial culture evenly on LB agar plates containing 100 μg / mL ampicillin (Amp) (if the plate density is low, it can be concentrated before spreading), and incubate at 37℃ for 12-16 h.

[0032] Screening and identification of E. coli positive clones: Five positive clones were picked from the plate and inoculated into 4 mL ampicillin culture tubes. The tubes were incubated overnight at 37°C and 220 rpm using a shaker. Plasmid DNA was extracted using a plasmid mini-extraction kit.

[0033] Plasmid extraction procedure: Take 4 mL of bacterial culture (centrifuge multiple times to remove supernatant), centrifuge at 12,000 ×g at room temperature for 1 min, and discard the supernatant. Add 250 μL of Buffer P1 (confirm RNase A is present before use) and vortex to resuspend. Add 250 μL of Buffer P2, gently vortex to mix 6-8 times, and incubate at room temperature for 2 min (no more than 5 min). Add 350 μL of Buffer N3, and immediately gently vortex to mix 6-8 times (avoid lysis of genomic DNA). Centrifuge at 13,000 ×g for 10 min at room temperature. Carefully transfer the supernatant to the adsorption column (multiple transfers are possible, but do not aspirate too much). Centrifuge at 12,000 ×g at room temperature for 1 min, and discard the filtrate. Add 750 μL of Buffer PE (confirm ethanol is present before use), centrifuge at 12,000 ×g for 1 min at room temperature, and discard the filtrate. Centrifuge again at 12,000 ×g for 3 min, open the cap and let stand for 10 min to remove residual ethanol and avoid contamination of downstream experiments. Place the adsorption column in a new 1.5 mL centrifuge tube, add 30 μL of Elution Buffer (EB) to the center of the adsorption column membrane, let stand at room temperature for 2 min, centrifuge at 13,000 × g for 1 min to collect the plasmid solution. If the plasmid concentration is low, the elution can be repeated multiple times.

[0034] 1.4 Screening and identification of Escherichia coli positive clones Five positive clones were picked from the plate and inoculated into 4 mL ampicillin culture tubes. The tubes were incubated overnight at 37°C and 220 rpm with shaking. Plasmid DNA was extracted using an alkaline lysis column-based plasmid mini-extraction kit. Plasmid concentration and purity were determined using NanoDrop (OD260 / 280 should be between 1.8 and 2.0).

[0035] XhoI enzyme was used for single-enzyme digestion identification. The enzyme digestion reaction system is shown in Table 1.3.

[0036] Table 1.3 Enzyme digestion reaction system

[0037] Configure the system according to the table above. After incubating at 37℃ for 2 hours, take 10 μL of the enzyme digestion product for 1% agarose gel electrophoresis for identification. If the ligation is successful, XhoI single enzyme digestion should produce a linear band of approximately 7706 bp. Select the clone with the correct digestion result for the next experiment. The nucleotide sequence of the PBE-cDNA4 plasmid vector is shown in SEQ ID NO:3.

[0038] Example 2 Construction and identification of the PBE-cDNA4-dsRNA plasmid vector: To construct a vector backbone capable of expressing dsRNA, this study used seamless cloning technology to insert a second inverted CMV promoter into the PstI site of pBE-cDNA4, forming a dual-promoter expression system pBE-cDNA4-dsRNA (SEQ ID NO:10). The final structure places the EcoRI and HindIII sites between two opposing CMV promoters.

[0039] 2.1 Cloning of the CMV promoter Using pBE-cDNA4 as a template, the CMV promoter was amplified by PCR using high-fidelity DNA polymerase. Primers were designed to ensure that the amplification product represented the inversely complementary CMV sequence, and to introduce arms (approximately 15-20 bp) at both ends that were homologous to the sequences flanking the PstI restriction site on pBE-cDNA4 for subsequent seamless cloning. The primer sequences are shown in Table 1.4.

[0040] Table 1.4 Primer sequences for CMV promoters (including homologous arms)

[0041] The PCR reaction system is shown in Table 1.5.

[0042] Table 1.5 PCR reaction system (high-fidelity enzyme)

[0043] After shaking and centrifuging the above system, the reaction was carried out on a PCR instrument under the conditions shown in Table 1.6.

[0044] Table 1.6 PCR reaction conditions

[0045] After amplification, 6 μL of the PCR product was taken for electrophoresis. Expected result: A specific band of approximately 253 bp should be obtained. The CMV promoter fragment was recovered and purified using a PCR product purification kit.

[0046] 2.2 Preparation of the pBE-cDNA4 linear vector The pBE-cDNA4 plasmid was linearized using the PstI restriction endonuclease (SEQ ID NO:17: CTGCAG). The enzyme digestion reaction system is shown in Table 1.7.

[0047] Table 1.7 Enzyme digestion reaction system

[0048] Prepare the system according to the table above, incubate at 37℃ for 2 hours, and take 3 μL of the enzyme digestion product for 1% agarose gel electrophoresis for identification. Expected result: A linear band of approximately 7.7 kb should be obtained. The linearized pBE-cDNA4 vector was recovered and purified using an agarose gel DNA recovery kit.

[0049] 2.3 Seamless cloning and ligation of the CMV promoter and the pBE-cDNA4 linear vector The purified CMV promoter PCR product was ligated to the linearized pBE-cDNA4 vector using seamless cloning technology. The reaction system is shown in Table 1.8.

[0050] Table 1.8 Seamless clonal linkage reaction system

[0051] Note: The molar ratio of vector to insert is 1:2; the volume is calculated based on their respective concentrations. Prepare the reaction system according to the table above, gently aspirate and mix, and briefly centrifuge. Incubate at 37°C for 30 min on a PCR instrument. After the reaction is complete, it can be used directly for conversion or stored at -20°C.

[0052] 2.4 Transformation of ligation products into Escherichia coli The method is the same as step 1.3 in this section. Coat it onto an LB solid plate containing Amp.

[0053] 2.5 Screening of E. coli positive clones Six single colonies were picked and inoculated into LB liquid medium containing Amp, and incubated overnight at 37°C and 220 rpm. Colony PCR verification was performed. Vector primers + insertion primers (forward primer t7, sequence SEQ ID NO:18: TAATACGACTCACTATAGG, reverse primer sequence SEQ ID NO:19: AGTCATCGCTATTACCATG), the PCR reaction system is shown in Table 1.9, and the reaction conditions are shown in Table 1.10.

[0054] Table 1.9 Colony PCR Reaction System

[0055] Table 1.10 PCR Reaction Conditions

[0056] After amplification, 6 μL of the PCR product was taken and the amplification results were checked by electrophoresis. The expected size was 300 bp.

[0057] Example 3 Obtaining RNA interference target sequences: 3.1 Extraction of total RNA Take approximately 20-50 mg of midgut or brain tissue from freshly dissected cotton bollworms, quick-frozen in liquid nitrogen, grind it into powder using liquid nitrogen, and perform the extraction step. Use NanoDrop to determine RNA concentration and purity (OD260 / 280 should be between 1.8 and 2.1, OD260 / 230 should be greater than 1.8).

[0058] Take 2 μL of RNA for 1% agarose gel electrophoresis and observe whether the ribosomal RNA bands (28S and 18S) are clear, intact, and in the correct ratio (approximately 2:1) to assess RNA integrity.

[0059] 3.2 Reverse transcription of RNA Take 1 μg of high-quality total RNA and reverse transcribe it using a full-length gold cDNA synthesis and gDNA removal kit. The entire experiment must be performed on ice. Add the solution from Table 1.11, shake well, and centrifuge for 5 seconds.

[0060] Table 1.11 Total RNA Reverse Transcription System

[0061] The reverse transcription reaction was performed on a PCR instrument.

[0062] 3.3 Amplification and purification of the AChE gene fragment Using cotton bollworm cDNA obtained through reverse transcription as a template, specific primers and high-fidelity DNA polymerase were used to amplify the AChE gene fragment required for constructing the RNA interference plasmid via PCR. The following fragments needed to be amplified: Fragment amplification for dsRNA construction: Primers were designed to amplify a 308 bp fragment from the core region of the AChE gene, with EcoRI and HindIII restriction sites added to the 5' end of the primers, respectively. The primer sequences are as follows: AChE-308-Fwd: 5'-GGAATTCCctcttagaccacataatgaactc -3', the nucleic acid sequence is shown in SEQ ID NO:8; AChE-308-Rev: 5'-CCCAAGCTTGGGgtggagactcaacgaagatc -3', the nucleic acid sequence is shown in SEQ ID NO:9; The Sense and Antisense fragments used for lhRNA construction (416 bp and 308 bp, respectively, with specific homologous arms) AChE-lh-Sense-Fwd:5'-ctagcgtttaaacttaagcttCTCTTAGACCACATAATGAACTCATATGA-3', the nucleic acid sequence is shown in SEQ ID NO:4; AChE-lh-Sense-Fwd: 5'-ttgagtctccacCCTTGAACCCAGGGAAGAATTA -3', the nucleic acid sequence is shown in SEQ ID NO:5; AChE-lh-Antisense-Fwd: 5'-gttcaaggGTGGAGACTCAACGAAGATCAATTG -3', the nucleic acid sequence is shown in SEQ ID NO:6; AChE-lh-Antisense-Fwd:5'-tgctggatatctgcagaattCCTCTTAGACCACATAATGAACTCATATGA -3', the nucleotide sequence is shown in SEQ ID NO:7.

[0063] For the amplification of the above three fragments (dsAChE fragment, lhAChE-Sense fragment, and lhAChE-Antisense fragment), high-fidelity DNA polymerase was used to reduce the error rate and ensure the efficiency of seamless cloning. The PCR reaction system is shown in Table 1.12, and the reaction conditions are shown in Table 1.13.

[0064] After amplification, 6 μL of PCR product was analyzed by 1% agarose gel electrophoresis. The expected dsAChE fragment was 308 bp, and the Sense and Antisense fragments of lhAChE were 416 bp and 308 bp, respectively. After confirming accurate band size and the absence of obvious nonspecific bands, the target fragment was purified using a DNA recovery kit. The concentration of the purified DNA fragment was determined using NanoDrop and stored at -20℃ for subsequent cloning reactions.

[0065] Table 1.12 PCR reaction system (high-fidelity enzyme)

[0066] Table 1.13 PCR reaction conditions (high-fidelity enzyme)

[0067] See the flowchart for constructing interference plasmids. Figure 3See the schematic diagrams of the two RNA interference plasmids. Figure 4 .

[0068] Example 4 PBE-cDNA4-lhAChE plasmid construction: This section describes the construction of the plasmid pBE-cDNA4-lhAChE, which expresses long hairpin RNA (lhRNA). Using seamless cloning technology, the linearized pBE-cDNA4 vector backbone, the AChE sense strand fragment (Sense, 416 bp) with specific homologous arms, and the AChE antisense strand fragment (Antisense, 308 bp) were assembled into an expression cassette containing the hairpin structure.

[0069] 4.1 Linearization of the pBE-cDNA4 vector The pBE-cDNA4 vector was double-digested with EcoRI and HindIII to produce a linearized vector backbone for seamless cloning. The digestion reaction system is shown in Table 1.14.

[0070] Table 1.14 pBE-cDNA4 vector double enzyme digestion reaction system

[0071] Prepare the digestion system according to the table above and incubate at 37°C for 2 hours. Detect the enzyme digestion efficiency by 1% agarose gel electrophoresis, and recover and purify the pBE-cDNA4 linearized vector using a gel recovery kit (approximately 7.6 kb in size). 4.2 Seamless Cloning Assembly of Three Fragments Table 1.15 Seamless clonal assembly reaction system

[0072] The purified linearized pBE-cDNA4 vector, Sense fragment, and Antisense fragment were mixed in an appropriate molar ratio (usually vector:Sense:Antisense ≈ 1:3:3) and the ligation reaction system for seamless cloning was shown in Table 1.15.

[0073] Prepare the system according to the table above, gently aspirate and mix, and briefly centrifuge. Place in a PCR instrument and react at 50°C for 30 minutes.

[0074] 4.3 Transformation and Screening Transform 10 μL of the assembly reaction product into TOP10 competent cells and plate them on LB agar plates containing Amp, following the same procedure as step 3 in section 1.1.1. After extracting plasmids from single colonies, digest them with EcoRI and HindIII.

[0075] Expected results: Positive clones should yield an lhRNA fragment of approximately 706 bp and a vector backbone band. Select the validated clone and name it pBE-cDNA4-lhAChE. See [link to pBE-cDNA4-lhAChE image] for the map of the recombinant plasmid vector pBE-cDNA4-lhAChE. Figure 1 The nucleotide sequence is shown in SEQ ID NO:1.

[0076] Example 5 PBE-cDNA4-dsAChE plasmid construction: Based on the PBE-cDNA4-dsRNA plasmid vector, this section utilizes the EcoRI and HindIII sites located between the two promoters to insert a 308 bp fragment of the AChE gene, constructing the plasmid pBE-cDNA4-dsAChE capable of simultaneously transcribing both sense and antisense RNA.

[0077] 5.1 Enzyme digestion of the vector Table 1.16 pBE-cDNA4-dsRNA vector enzyme digestion reaction system (EcoRI / HindIII)

[0078] The pBE-cDNA4-dsRNA plasmid vector was double-digested with EcoRI and HindIII to linearize the vector while exposing the cloning site located between the two inverted CMV promoters. The pBE-cDNA4-dsRNA vector digestion reaction system is shown in Table 1.16.

[0079] Reaction conditions: 37℃ water bath for 4 hours. Take 5 μL of the digestion product and use 1% agarose gel electrophoresis to detect the digestion effect. The band size should be approximately 7.9 kb (linearized PBE-cDNA4-dsRNA plasmid vector). Purify the linearized vector using a gel extraction kit.

[0080] 5.2 Seamless cloning of plasmids The AChE target sequence fragment (308 bp) amplified and purified in Section 1.1.3, with homologous arms at both ends to the vector ends of the EcoRI / HindIII restriction sites, was ligated into the linearized pBE-cDNA4-dsRNA vector using seamless cloning technology. The seamless cloning reaction system is shown in Table 1.17.

[0081] Table 1.17 Seamless Cloning Reaction System

[0082] Configure the system according to the table above, gently aspirate and mix, briefly centrifuge, and then place in a PCR machine at 37°C for 30 min. After the reaction, remove and store in a -20°C freezer or use directly for conversion.

[0083] 5.3 Transformation and Identification of Plasmids 10 μL of the seamless cloning product was transformed into competent cells and plated on LB agar plates containing ampicillin. The plates were incubated overnight at 37°C. Five positive clones were picked and seeded into 4 mL LB culture tubes containing ampicillin. The cells were incubated overnight at 37°C with shaking at 220 rpm. Plasmid DNA was extracted using a plasmid mini-extraction kit and double-digested with EcoRI and HindIII. The reaction system is shown in Table 1.16. Expected results: Positive clones should yield approximately 308 bp of AChE insert and approximately 7.9 kb of vector backbone. All final recombinant plasmids (pBE-cDNA4, pBE-cDNA4-dsRNA, pBE-cDNA4-lhAChE, pBE-cDNA4-dsAChE) were finally verified by Sanger sequencing to ensure correct insertion and ligation. The pBE-cDNA4-dsAChE pattern is shown in [reference needed]. Figure 2 .

[0084] The nucleotide sequence of the recombinant plasmid pBE-cDNA4-dsAChE is shown in SEQ ID NO:2.

[0085] Experimental Example 1 Two plasmids (dsAChE plasmid and lhAChE plasmid) were introduced into the cotton bollworm through feeding, and the physiological state of the cotton bollworm was observed and recorded to verify the effectiveness of biological control.

[0086] The cotton bollworms (Helicoverpa armigera) used in the experiment were from a laboratory population preserved by Keyuan Biotechnology Co., Ltd. This population had been continuously reared indoors for over 60 generations without exposure to any pesticides. Rearing conditions were: temperature 27±1℃, relative humidity 70±5%, and photoperiod 16L:8D. Larvae were reared on artificial feed (main components: 25% corn flour, 15% soybean flour, 8% yeast powder, 5% vitamin mixture, 2% agar, and the remainder water).

[0087] Feed preparation: The purified RNAi plasmid (lhAChE or dsAChE) or empty vector was diluted to 100 ng / μL with RNase-free PBS. A piece of artificial feed (approximately 0.5 cm³) was evenly coated with 10 μL of plasmid solution (containing 1 μg of plasmid) and air-dried at room temperature for approximately 10 min to allow the solution to adsorb. The blank control group was treated with an equal volume of PBS.

[0088] Feeding experiment: Cotton bollworm larvae at the first, second, and third instars were fed once daily (the amount of food was a small piece from the prepared feed), and the feed was replaced with fresh feed daily. Individual larvae were placed in rearing boxes containing the corresponding treated feed. Treatment groups (lhAChE, dsAChE), a negative control group (empty vector), and a blank control group (PBS) were established.

[0089] 1. Effect of plasmid feeding on the survival rate of cotton bollworm larvae Feed preparation: The purified RNAi plasmid (lhAChE or dsAChE) or empty vector was diluted to 100 ng / μL with RNase-free PBS. Approximately 0.5 cm³ of artificial feed was taken, and 10 μL of plasmid solution (containing 1 μg of plasmid) was evenly spread on its surface. The feed was then air-dried at room temperature for approximately 10 min to allow the solution to adsorb. The blank control group was treated with an equal volume of PBS.

[0090] To investigate the effect of RNAi plasmids on the survival rate of cotton bollworms, lhAChE plasmids and dsAChE plasmids targeting acetylcholinesterase (AChE) were fed to first-instar, second-instar, and third-instar larvae, respectively. An empty plasmid group and a conventional feed control group were set up. The observation continued until the pupal stage, and the cumulative mortality rate, weight change rate, and pupation rate were recorded.

[0091] The lethality of RNAi exhibits a strong age-dependent effect. For example... Figure 5 The mortality rate decreased with increasing starting age of feeding (from one to three years old), while the mortality rates of the lhAChE plasmid and dsAChE plasmid groups were significantly higher than those of the control group. The mortality rate of the lhAChE plasmid group was the highest (69.67±5.51%), which was significantly higher than that of the dsAChE plasmid group (57.33±0.58%) (P<0.05).

[0092] Table 2. Comparison of mortality rates of cotton bollworms at different ages from feeding with various RNAi plasmids to the pupal stage.

[0093] Table 2 shows that the mortality rates of the two experimental groups were significantly higher than those of the control group and the empty plasmid group, and decreased with age.

[0094] Table 3. Comparison of pupal weights of surviving individuals of *Bollworm* in different RNAi plasmid treatment groups.

[0095] Note: ** indicates P < 0.01 Table 3 shows that in surviving individuals, the lhAChE plasmid and dsAChE plasmid groups also had significant effects, reducing body weight and greatly decreasing the cotton bollworm's ability to pupate in the wild, thereby reducing its reproductive capacity and achieving the effect of prevention and control.

[0096] This study verified the lethality of RNAi plasmids administered orally to cotton bollworms, and crucially revealed the negative correlation between RNAi efficiency and larval instar, comparing the differences in efficacy between different plasmid constructions. The results clearly demonstrate the significant role of RNAi technology in controlling lepidopteran pests such as cotton bollworms, and validate that the focus of control should be placed on the early larval stages (especially the first instar) to achieve the best control effect.

[0097] Experiment Example 2 Distribution of RNA interference plasmids in cotton bollworms: To investigate the distribution characteristics of RNA interference plasmids in cotton bollworms and their relationship with RNAi effects, this study used qPCR to quantitatively detect the content and dynamic changes of the lhAChE plasmid (a representative RNA interference plasmid) in four major tissues of cotton bollworms. The results revealed the distribution patterns of RNA interference plasmids in insects, providing important evidence for optimizing the application of RNA interference technology in agricultural pest control.

[0098] qPCR data were statistically analyzed and subjected to one-way ANOVA using GraphPad Prism 10. A p-value < 0.05 indicated a significant difference in gene expression levels, and a p-value < 0.01 indicated a highly significant difference.

[0099] Data analysis was performed using GraphPad Prism 10 or similar statistical software. Two-way ANOVA was used to examine the effects of tissue type and time point, and their interaction, on plasmid copy number (log transformation may be required to ensure homogeneity of variance). If the ANOVA results were significant, Tukey's or Sidak's multiple comparison tests were further used to analyze the specific differences between different tissues or different time points. Significance levels were set at P < 0.05 (*) and P < 0.01 (**).

[0100] ANOVA results showed that tissue type had a significant impact on the distribution of lhAChE plasmid in vivo (F(3, 32) = 3.528, P = 0.0258). As shown in Figure 6, at 48 h post-feeding, the distribution of the plasmid in different tissues followed the pattern of midgut > fat body > hemolymph > brain.

[0101] Figure 6 This indicates that orally delivered lhAChE plasmids are unevenly distributed within the cotton bollworm, primarily enriched in the midgut tissue of direct contact (peak concentration approximately 3.6 × 10⁻⁶). 4 The highest concentration of plasmids was found in the midgut (copies / mg wet weight), followed by metabolically active fat bodies, while its concentration was significantly lower in hemolymph and the brain. This distribution pattern is determined by the physiological structure of the insect digestive tract, the characteristics of the circulatory system, and the physicochemical properties of the plasmids themselves. It should be noted that although the midgut tissue had the highest relative plasmid concentration, its absolute value was still lower than that of highly efficient delivery systems (such as nano / viral vectors). Furthermore, the total plasmid DNA detected by qPCR includes extracellular attachment, cell membrane binding, and intracellular delivery components. Given the inherent cell membrane penetration barrier of naked plasmid DNA, the proportion of plasmids that actually enter the cell and possess transcriptional function may be extremely low. This limitation in delivery efficiency is one of the key bottlenecks in the application of orally administered plasmid RNAi.

[0102] Implications for optimizing RNAi strategies: Helicobacter pylori nucleopolyhedrovirus (HaNPV), as a natural insect pathogen, possesses the ability to efficiently infect Helicobacter pylori cells (especially the midgut). Its infection process involves steps such as disrupting the midgut peritrophic membrane, fusing with the cell membrane, or endocytosis into the cell. These characteristics not only endow HaNPV with insecticidal potential but also provide a potential pathway for enhancing the entry of exogenous molecules (such as RNAi plasmids) into cells.

[0103] Experiment Example 3: Combined application of RNA interference plasmid and cotton bollworm nucleopolyhedrovirus (HaNPV) Feeding studies have shown that the distribution of RNA interference plasmids within cotton bollworms presents a cell entry barrier, limiting the effectiveness of RNA interference. Viruses, as highly efficient gene delivery systems in nature, possess the unique ability to breach cell membrane barriers. This chapter aims to explore the synergistic effect of combined application of RNA interference plasmids and cotton bollworm nucleopolyhedrovirus (HaNPV), achieving efficient control of cotton bollworms through the complementary advantages of these two biopesticides.

[0104] Bollworm nucleopolyhedrovirus (HaNPV) is a baculovirus that specifically parasitizes the bollworm. It is highly specific to the target pest and safe for humans, animals, and the environment. However, HaNPV suffers from slow onset and unstable efficacy in field applications, limiting its widespread use. As a cell invasion "expert," HaNPV may help RNA interference plasmids enter target cells more effectively, thereby improving RNA interference efficiency. Based on this, the following questions will be studied: (1) the lethal effect of combined application of RNA interference plasmids and HaNPV on bollworms; (2) whether RNA interference can enhance HaNPV infection and replication; (3) whether HaNPV can improve the delivery efficiency of RNA interference plasmids; and (4) the optimal strategy and suitable conditions for combined application. These studies will provide a scientific basis for developing more efficient biopesticide combinations and are of great significance for the biological control of bollworms.

[0105] Experimental materials and equipment Experimental insects: The cotton bollworms (Helicoverpa armigera) used in the experiment were from a laboratory population preserved by Keyuan Biotechnology Co., Ltd. This population had been continuously reared indoors for over 60 generations without exposure to any pesticides. Rearing conditions were: temperature 27±1℃, relative humidity 70±5%, and photoperiod 16L:8D. Larvae were reared in an artificial diet (main components: 25% corn flour, 15% soybean flour, 8% yeast powder, 5% vitamin mixture, 2% agar, and the remainder water), while adults were reared in a 10% honey solution. Healthy, uniformly developed early-stage 3rd instar larvae (weighing 35±3 mg) were selected and first starved for 4 hours before being used in each treatment group.

[0106] RNA interference plasmid: The lhAChE plasmid, dsAChE plasmid, and empty plasmid (plastic mid-gene not inserted) used in this study were successfully constructed and stored at -80°C as described above.

[0107] Cotton bollworm nucleopolyhedrovirus (HaNPV): The bollworm nucleopolyhedrovirus (HaNPV) used in the experiment was provided by Keyuan Biotechnology Co., Ltd., with an initial titer of 4 × 10^8 PIB / mL (polyhedrome inclusion bodies / mL). Before use, the virus stock solution was diluted with sterile PBS (pH 7.4) to a working concentration of 5 × 10^7 PIB / mL. The virus suspension was stored at 4°C in the dark, avoiding repeated freeze-thaw cycles. Viral activity was validated periodically through bioassays on standard susceptible bollworm populations to ensure stable efficacy.

[0108] Processing group settings: According to the experimental objective, the following 6 treatment groups were set up, with 30 early third instar cotton bollworm larvae in each group, and the experiment was repeated 3 times: the larvae were randomly divided into 6 groups, with 30 larvae in each group, and the experiment was repeated 3 times. According to the treatment design, the appropriate concentration of HaNPV suspension and / or RNA interference plasmid solution was mixed and incubated for 15 minutes. Then, the mixture was evenly spread on the surface of 1 cm³ artificial feed (total volume not exceeding 50 μL), dried at room temperature for 30 minutes, and then fed to the larvae. They were fed once a day as in Experiment 1, and their size was changed to 1 cm³.

[0109] Control group: fed an equal volume of sterile PBS (pH 7.4) treated artificial feed; HaNPV treatment group: fed artificial feed containing HaNPV (5×10^7 PIB / mL); lhAChE treatment group: fed artificial feed containing long hairpin RNA expression plasmid (1 μg / μL); dsAChE treatment group: fed artificial feed containing double-stranded RNA expression plasmid (1 μg / μL); lhAChE+HaNPV combined treatment group: fed with artificial feed containing equal amounts of long hairpin RNA expression plasmid (1 μg / μL) and HaNPV (5×10^7 PIB / mL); dsAChE+HaNPV combined treatment group: The artificial feed containing equal amounts of double-stranded RNA expression plasmid (1 μg / μL) and HaNPV (5×10^7 PIB / mL) was treated by feed mixing.

[0110] First, following the treatment design, the appropriate concentration of HaNPV suspension and / or RNA interference plasmid solution was mixed and incubated for 15 minutes. The mixture was then evenly spread on a 1 cm³ surface of artificial feed (total volume not exceeding 50 μL), dried at room temperature for 30 minutes, and then fed to the larvae. Each larva was individually reared in a 12-well plate. After 24 hours of treatment, the larvae were replaced with normal artificial feed and reared. Throughout the experiment, the feed was changed daily, feces were removed, and the culture environment was kept clean. The culture conditions were the same as the rearing conditions: temperature 27±1℃, relative humidity 70±5%, and photoperiod 16L:8D.

[0111] Indicator measurement methods: Mortality statistics: For five consecutive days following treatment, larval mortality was observed every 24 hours. Mortality was determined by: no reaction upon gentle touch of the larvae's body surface with a dissecting needle, and a noticeable change in body color (fading or blackening). The number of dead larvae in each group was recorded daily, and the cumulative mortality rate was calculated. Cumulative mortality rate (%) = (cumulative number of dead larvae / initial total number of larvae) × 100%.

[0112] Weight measurement: Using an electronic balance with an accuracy of 0.1 mg, the weight of larvae in each group was measured before treatment (day 0) and on days 2, 3, 4, and 5 after treatment. Before each measurement, the larvae were gently wiped clean to remove surface feed and fecal residue, and then placed in pre-weighed petri dishes for weighing. Ten larvae were randomly selected from each group for measurement, and the measurements were repeated three times. The average value was calculated.

[0113] RNA extraction and qRT-PCR analysis: a. Sample collection and processing After 72 hours of treatment, six larvae were randomly selected from each group, flash-frozen in liquid nitrogen, and stored at -80°C. Before RNA extraction, three larvae from each group were combined into one biological replicate, for a total of two biological replicates.

[0114] b. Total RNA extraction Total RNA was extracted using TRIzol reagent. The procedure was the same as described above. RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer, and RNA integrity was checked by agarose gel electrophoresis.

[0115] c. cDNA synthesis Take 500 ng of qualified total RNA and perform reverse transcription using a full-gold cDNA synthesis and gDNA removal kit. Set the program for the reverse transcription reaction (see Section 2.2.3, Step 2 for detailed steps). Store the synthesized cDNA at -20℃.

[0116] d. Real-time quantitative PCR (qRT-PCR) qRT-PCR analysis was performed using TransStart® Green qPCR SuperMix UDG, with the β-actin gene used as an internal control. Primer sequences are as follows: Table 4.6 Primer sequences for the target gene AChE and the internal reference gene β-actin

[0117] Reaction system (20 μL): Table 4.7 qPCR reaction system

[0118] The qPCR amplification conditions are shown in Table 4.8: Table 4.8 qPCR amplification conditions.

[0119]

[0120] Melting curve analysis (65~95℃, increasing by 0.5℃ per step) was performed to verify amplification specificity.

[0121] Three technical replicates were set up for each sample, and a template-free control (NTC) was also set up.

[0122] e. Data Analysis The relative expression levels of target genes were calculated using the 2^(-ΔΔCT) method:

[0123]

[0124]

[0125]

[0126] Synergy Index (CI) Calculation The Chou-Talalay method was used to calculate the synergistic effect index (CI) between RNA interference plasmids and HaNPV:

[0127] Where D1 and D2 are the doses of the two agents in the combined treatment, respectively; Dx1 and Dx2 are the doses required to produce the same effect as the combined treatment when used alone.

[0128] CI value interpretation criteria: CI < 0.9 indicates a synergistic effect, 0.9 ≤ CI ≤ 1.1 indicates an additive effect, and CI > 1.1 indicates an antagonistic effect. The smaller the CI value, the stronger the synergistic effect; Pathological changes observation: Observation method: Use a dissecting microscope to observe changes in the external morphology of the larvae; Record content: Changes in body color, body shape, activity level, feeding behavior, etc.; Image acquisition: Use a digital camera to record typical pathological changes; Data processing and statistical analysis: All data were statistically analyzed using SPSS 26.0 software. Quantitative data are expressed as mean ± standard error (Mean ± SE). The standard error (SE) of the expected values ​​was calculated using the independent samples error propagation formula:

[0129] Multiple comparisons among groups were performed using one-way ANOVA and the least significant difference (LSD) test, with a significance level set at P < 0.05. Independent samples t-tests were used to compare the treatment group with the control group or the theoretical cumulative values.

[0130] Mortality data were transformed using the arcsine square root method before statistical analysis. Gene expression data were log-transformed to meet the requirements of normal distribution before statistical analysis. All charts were created using GraphPad Prism 8.0 software.

[0131] Theoretical superposition value calculation To assess whether the combined treatment has a synergistic effect, the theoretical superposition value of the individual treatments was calculated:

[0132]

[0133] EA and EB represent the effect values ​​when using the RNA interference plasmid and HaNPV alone, respectively. WA, WB, and WControl represent the body weights of the user using the RNA interference plasmid and HaNPV, and the control group, respectively, at the same time point.

[0134] Results and Discussion Dynamic changes in cumulative larval mortality rates in each treatment group: Table 4.9 Cumulative mortality rate of bollworm larvae in different treatment groups (%)

[0135] Note: Data are mean ± standard error, ** indicates P<0.01, compared with theoretical superposition values.

[0136] Mortality data showed that the cumulative 5-day mortality rate was 30.0% in the HaNPV monotherapy group and 10.0% in both the lhAChE and dsAChE monotherapy groups. The combined treatment groups showed a synergistic effect as early as day 3: the mortality rate in the lhAChE+HaNPV group reached 41.3%, significantly higher than the sum of the single treatment groups (10.0%); by day 5, the mortality rate in the lhAChE+HaNPV group rose to 72.4%, and in the dsAChE+HaNPV group to 62.0%, both significantly exceeding the theoretical values ​​of the independent-effect model (30.0%+10.0%-30.0%×10.0%=37.0%) (P<0.01). See [link to relevant documentation]. Figure 7 .

[0137] As shown in Table 4.1, the survival curves of the combined treatment groups exhibited a characteristic rapid attenuation: the survival rate of the lhAChE+HaNPV group (Table 4.1A) dropped sharply from day 3, with a 5-day survival rate of only 27.6%; the dsAChE+HaNPV group (Table 4.1B) showed a similar trend but with a slightly lower intensity (5-day survival rate of 38.0%). This rapid knockdown effect can effectively reduce crop damage. Notably, the combination of lhAChE (long hairpin RNA) and HaNPV was more effective than dsAChE (double-stranded RNA), which may be related to the structural stability of lhRNA in insects.

[0138] Survival curve analysis further confirmed that the combined treatment group not only had a higher final mortality rate, but more importantly, its mortality process was significantly accelerated. The combined treatment group showed a faster onset of death and a steeper slope of the survival curve. In particular, the lhAChE+HaNPV group showed a sharp drop in survival rate from 58.7% to 38.0% within 24 hours from day 3 to day 4, demonstrating a strong "rapid knockout" effect.

[0139] In the combined treatment, the combination of long hairpin RNA (lhAChE) and HaNPV was more effective than the combination of double-stranded RNA (dsAChE) and HaNPV. This may be related to the fact that lhRNA has a more stable structure, degrades more slowly in larvae, and is more efficient at inducing RNA interference.

[0140] Synergy Index (CI) Analysis To quantitatively assess the interaction between the two biologics, we calculated the synergistic effect index (CI): Table 4.10 Synergistic effect index of RNA interference plasmid combined with HaNPV

[0141] According to the CI evaluation criteria, CI < 0.9 indicates a synergistic effect, 0.9 ≤ CI ≤ 1.1 indicates an additive effect, and CI > 1.1 indicates an antagonistic effect. The CI values ​​of both combined treatments were significantly less than 0.9, confirming a significant synergistic effect between the RNA interference plasmid and HaNPV, with the lhAChE+HaNPV group (CI = 0.53) showing a stronger synergistic effect than the dsAChE+HaNPV group (CI = 0.64).

[0142] The existence of this synergistic effect means that in practical applications, the combined use of RNA interference plasmids and HaNPV can significantly reduce the dosage of each component while maintaining a high control effect, thereby reducing production costs and environmental burden. At the same time, the rapid insecticidal effect of the combined use also helps improve the timeliness of control, especially during pest outbreaks.

[0143] Effects of combined treatment with RNA interference plasmid and HaNPV on the growth and development of cotton bollworm larvae: Larval weight monitoring showed that the control group gained approximately 4.4 times its normal weight within 5 days (35.6 mg → 156.3 mg). The HaNPV-only treatment group (105.6 mg) had a 32.4% lower weight than the control group, while the lhAChE (127.8 mg) and dsAChE (128.4 mg)-only treatment groups only showed reductions of 18.2% and 17.8%, respectively. The combined treatments produced a significant synergistic inhibitory effect: the lhAChE+HaNPV group (67.5 mg) had a 56.8% lower weight than the control group, and the dsAChE+HaNPV group (73.2 mg) had a 53.2% lower weight. The key finding was that from day 2 of treatment, the combined group's weight was consistently significantly lower than the theoretical sum of the individual treatments (P < 0.05), confirming the early effectiveness of the synergistic effect. This growth inhibition effect corroborated the mortality data, indicating that the physiological functions of surviving larvae were also severely impaired. In agricultural practice, this continuous growth and feeding inhibition can directly reduce the intensity of pest damage, achieving the dual control goals of "lethal control" and "damage inhibition".

[0144] Table 4.11 Changes in body weight of cotton bollworm larvae in different treatment groups (mg)

[0145] Note: Data are presented as mean ± standard error (n=30). ** indicates a significant difference compared to the theoretical additive effect (P<0.01). Effects of combined treatment with RNA interference plasmid and HaNPV on the pathology and physiology of cotton bollworm larvae: Larvae in the combined treatment group exhibited significantly aggravated typical pathological symptoms. Three days after treatment, both the lhAChE+HaNPV and dsAChE+HaNPV groups showed: body color changing from normal green / brown to pale or light yellow, thinning and fragility of the body wall, arched body, sluggish movement and dulled response to stimuli, accompanied by significant anorexia. Before death, they characteristically displayed the "V"-shaped posture (head raised) marked by baculovirus infection. In contrast, the HaNPV-only treatment group only showed mild body color lightening and reduced activity; the RNAi plasmid-only treatment group showed no significant external abnormalities. This pathological difference confirms that the combined treatment not only accelerates the viral infection process but also induces comprehensive functional impairment through interference with multiple physiological pathways. This early pathological phenotype is highly consistent with mortality and growth inhibition data, jointly confirming the rapid-acting characteristics of the combined treatment and providing key empirical evidence for developing rapid and efficient biocontrol strategies. See the typical pathological symptoms of the combined treatment group larvae for illustration. Figure 8 .

[0146] Target gene expression analysis To elucidate the molecular mechanism of the combined treatment, the expression dynamics of the target gene acetylcholinesterase (AChE) were analyzed by qRT-PCR. Figure 9 The results show: lhAChE group ( Figure 9 A): After 72 hours of treatment, the relative expression level of AChE decreased to 0.49 (inhibition rate 51.0%). lhAChE+HaNPV group ( Figure 9 A): The expression level further decreased to 0.10 (inhibition rate 90.0%), which was significantly lower than that of lhAChE treatment alone (P<0.01). dsAChE group ( Figure 9 B): Expression level was 0.48 (inhibition rate 52.0%). dsAChE+HaNPV group ( Figure 9 B): The expression level decreased to 0.15 (inhibition rate 85.0%), which was also significantly better than dsAChE single agent (P<0.01). HaNPV single-dose group ( Figure 9 B) No significant effect on AChE expression (relative expression level 0.90, inhibition rate 10.0%).

[0147] The above data confirm that HaNPV can significantly enhance the gene silencing efficiency of RNAi plasmids, and the combined treatment increases the AChE transcriptional repression rate by 35-39 percentage points.

[0148] By comparing the two subplots, it can be found that the lhAChE+HaNPV group showed a stronger gene suppression effect than the dsAChE+HaNPV group (90.0% vs 85.0%), which is consistent with the mortality data, further confirming that long hairpin RNA (lhAChE) may have better stability and the ability to induce RNA interference in insects than double-stranded RNA (dsAChE).

[0149] This significant gene expression suppression supports our hypothesis that HaNPV may enhance the delivery efficiency of RNA interference plasmids. The high level of AChE suppression, a key enzyme in the insect nervous system, directly explains the neurological dysfunction symptoms and higher mortality rates exhibited by larvae in the combined treatment group. When AChE expression is suppressed, acetylcholine accumulates in the synaptic cleft, leading to persistent nerve impulses and ultimately causing paralysis and death in the insects. The significantly enhanced gene suppression effect observed in the combined treatment group provides molecular-level evidence for understanding its synergistic insecticidal mechanism.

[0150] Results analysis: Experiment 3 systematically evaluated the synergistic control effect of RNA interference (RNAi) plasmid combined with HaNPV on cotton bollworm by measuring mortality, body weight change, and target gene (AChE) expression levels. The results showed that: Synergistic lethal effect: After 5 days of treatment, the larval mortality rates of the lhAChE+HaNPV and dsAChE+HaNPV combined treatment groups reached 72.4% and 62.0%, respectively, which were significantly higher than the expected summative effect of each individual treatment group (*p*<0.05), and the synergistic lethal effect was already apparent after 3 days of treatment.

[0151] Growth and development inhibition: The combined treatment group showed significant inhibition of larval feeding and weight gain, with the average weight reduced by more than 50% after 5 days of treatment compared to the control group.

[0152] Enhanced RNAi efficiency: After 72 hours of treatment with the combination therapy, the expression level of AChE gene decreased by 85-90%, while the RNAi treatment alone only inhibited it by about 50% (*p*<0.01), confirming that HaNPV can significantly improve the intracellular delivery efficiency of RNAi plasmids.

[0153] Vector form differences: lhAChE (long hairpin RNA) combined with HaNPV was more effective than dsAChE (double-stranded RNA), indicating that lhRNA may have higher stability in insects.

[0154] Table of synergistic effects analysis of RNAi plasmids and HaNPV

[0155] The results of this study provide a scientific basis for the development of novel compound biological pesticides: 1. Overcoming individual limitations: Combined application can effectively overcome the problems of slow onset and unstable efficacy of HaNPV, while solving the cell entry barrier of RNA interference plasmids, achieving complementary advantages. As the aforementioned mortality data shows, combined treatment can significantly accelerate the insecticidal effect, achieving a high mortality rate after 3 days of treatment. 2. Potential for reduced dosage: Due to the significant synergistic effect, it may be possible to reduce the dosage of each individual component in practical applications while maintaining a high control effect and reducing production costs. 3. Environmentally friendly control strategy: Both biological agents have high target specificity and environmental safety, and their combined application provides a new approach for developing green, environmentally friendly, and efficient cotton bollworm control technologies.

[0156] The above description of the embodiments and experimental examples is to enable those skilled in the art to understand and use the invention. The preparation and application of lhAChE plasmid and dsAChE plasmid control the growth and reproduction of cotton bollworm through RNA interference technology, and can be synergistically assisted by virus (HaNPV). It can serve as a novel biological control tool, suitable for the biological control of agricultural pests, and can increase crop yield by reducing the use of chemical pesticides, thus meeting the needs of green agriculture.

Claims

1. A double-stranded circular DNA, characterized in that: The double-stranded circular DNA includes recombinant plasmids pBE-cDNA4-lhAChE and pBE-cDNA4-dsAChE; wherein... The nucleotide sequence of the recombinant plasmid pBE-cDNA4-lhAChE is shown in SEQ ID NO:1; The nucleotide sequence of the recombinant plasmid pBE-cDNA4-dsAChE is shown in SEQ ID NO:

2.

2. The double-stranded circular DNA as described in claim 1, characterized in that: The double-stranded circular DNA efficiently expresses long hairpin RNA1 or double-stranded RNA in cotton bollworms. The DNA carries the sense and antisense sequences of the target gene, forms a double-stranded RNA structure, and is stably expressed.

3. The double-stranded circular DNA as described in claim 1 or 2, characterized in that: The double-stranded circular DNA is adapted to the promoter of the cotton bollworm to promote efficient expression within the host cell.

4. The method for preparing double-stranded circular DNA according to any one of claims 1 to 3, characterized in that: include, Construct hairpin structures or double-stranded RNA structures targeting cotton bollworm gene targets; The hairpin structure was integrated into a covalently closed circular DNA vector to obtain pBE-cDNA4-lhAChE and recombinant plasmid; The double-stranded RNA structure was integrated into a covalently closed circular DNA vector to obtain the pBE-cDNA4-dsAChE recombinant plasmid. The DNA vector is pBE-cDNA4, and the nucleotide sequence of pBE-cDNA4 is shown in SEQ ID NO:

3.

5. The preparation method according to claim 4, characterized in that: The integration of the hairpin structure into a covalently closed circular DNA vector includes, Using seamless cloning technology, the linearized pBE-cDNA4 vector backbone, the AChE sense strand fragment Sense with specific homologous arms, and the AChE antisense strand fragment Antisense were assembled into an expression cassette containing a hairpin structure. The AChE justice chain fragment is 416bp: AChE-lh-Sense-Fwd:5'-ctagcgtttaaacttaagcttCTCTTAGACCACATAATGAACTCATATGA-3', the nucleotide sequence is shown in SEQ ID NO:4; AChE-lh-Sense-Fwd: 5'-ttgagtctccacCCTTGAACCCAGGGAAGAATTA -3', nucleotide sequence as shown in SEQ ID NO:5; Among them, the AChE antisense fragment is 308 bp: AChE-lh-Antisense-Fwd: 5'-gttcaaggGTGGAGACTCAACGAAGATCAATTG -3', nucleotide sequence as shown in SEQ ID NO:6; AChE-lh-Antisense-Fwd:5'-tgctggatatctgcagaattCCTCTTAGACCACATAATGAACTCATATGA -3', the nucleotide sequence is shown in SEQ ID NO:

7.

6. The preparation method according to claim 4, characterized in that: The integration of the double-stranded RNA structure into a covalently closed circular DNA vector includes, The PBE-cDNA4-dsRNA plasmid vector was used to insert a 308 bp fragment of the AChE gene into the EcoRI and HindIII sites located between the two promoters, thus constructing the plasmid pBE-cDNA4-dsAChE, which can simultaneously transcribe the sense and antisense strands of RNA. The primer sequences for amplifying the 308 bp fragment of the AChE gene are as follows: AChE-308-Fwd: 5'-GGAATTCCctcttagaccacataatgaactc -3', nucleotide sequence as shown in SEQ ID NO:8; AChE-308-Rev: 5'-CCCAAGCTTGGGgtggagactcaacgaagatc -3, nucleotide sequence as shown in SEQ ID NO:9; The nucleotide sequence of the PBE-cDNA4-dsRNA plasmid vector is shown in SEQ ID NO:

10.

7. The application of the double-stranded circular DNA as described in claim 1 or 2 in the biological control of cotton bollworm.

8. The application as described in claim 7, characterized in that: This includes delivery of the virus into the cotton bollworm via feeding or a combination of feeding and administration, inducing the silencing of target genes and inhibiting the growth and development of the cotton bollworm.

9. A composition for inhibiting the growth and development of cotton bollworms, characterized in that: This includes the cotton bollworm nucleopolyhedrovirus HaNPV and the double-stranded circular DNA described in any one of claims 1 to 3, which synergistically inhibit the growth and development of cotton bollworms.

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