RNA biological pesticide targeting HcAmy2 gene of hyphantria cunea and preparation method thereof

Abstract

This invention discloses an RNA biopesticide targeting the HcAmy2 gene of the fall webworm and its preparation method, relating to the fields of biopesticides and green pest control technology. The active ingredient of the biopesticide is double-stranded RNA targeting the α-amylase gene of the fall webworm, namely dsHcAmy2, and is encapsulated by chitosan to form a chitosan / dsRNA nanocomposite. The degree of deacetylation of the chitosan is ≥95%, and the viscosity is 100-200 mPa·s. The nanoparticles prepared by this invention can effectively protect dsRNA from degradation, significantly enhance its stability and RNA interference efficiency, effectively silence the key gene HcAmy2 of the fall webworm, destroy its multi-host adaptability, and have a highly efficient and specific control effect on fall webworm larvae.

Description

Technical Field

[0001] This invention relates to the field of biological pesticides and green pest control technology, specifically to an RNA biological pesticide targeting the HcAmy2 gene of the fall webworm and its preparation method. Background Technology

[0002] The fall webworm (Hyphantria cunea) is a global quarantine pest with a diverse diet, causing significant damage and spreading rapidly, posing a serious threat to my country's forestry and ecological security. Currently, the control of the fall webworm still relies mainly on chemical pesticides, but long-term use has led to increasingly prominent problems such as increased pesticide resistance in pests, environmental pollution, and disruption of the ecological balance.

[0003] To address the aforementioned drawbacks of chemical pesticides, RNA interference (RNAi) technology, with its high species specificity and good environmental compatibility, has become an important direction for the research and development of next-generation green pesticides. This technology, by introducing double-stranded RNA (dsRNA) homologous to the target gene, can specifically silence key functional genes in pests, thereby inhibiting their growth and development or directly causing death, and is less likely to induce pesticide resistance.

[0004] However, the application of naked dsRNA as a pesticide faces significant technical bottlenecks: on the one hand, dsRNA has extremely poor stability, easily degraded by ultraviolet radiation and rainwater in the natural environment, and rapidly hydrolyzed by nucleases in the midgut fluid of insects, resulting in insufficient effective doses reaching target tissues and low RNAi interference efficiency; on the other hand, the duration of action of naked dsRNA is extremely short, usually only lasting a few hours, requiring frequent application to achieve control effects, thus limiting its practical application in the field. Furthermore, in existing RNAi pesticide development, some schemes have not fully considered the biocompatibility of carrier materials, and the selected synthetic carriers may have problems such as difficult degradation and potential ecotoxicity, further hindering technology transfer.

[0005] In addition to the mainstream technologies mentioned above, traditional biological pesticides (such as Bacillus thuringiensis and nucleopolyhedrovirus) are environmentally friendly, but they have drawbacks such as a narrow host range, significant influence of environmental conditions such as temperature and humidity on control effectiveness, and slow onset of action, making them difficult to meet the urgent control needs of large-scale outbreaks of fall webworm. Physical control methods (such as manual removal of webs and light trapping) have problems such as low efficiency, high labor intensity, and limited applicability, and are only suitable for small-scale, low-density pest outbreaks, and cannot meet the needs of large-scale control.

[0006] Meanwhile, the American white moth has a strong multi-host adaptability and can switch between different host plants with different preferences, such as ash, birch, and linden. Existing control technologies are mostly designed for single host scenarios and lack targeted solutions for its multi-host adaptability, resulting in unstable control effects. Summary of the Invention

[0007] The purpose of this invention is to provide an RNA biopesticide targeting the HcAmy2 gene of the fall webworm and its preparation method, thereby addressing the drawbacks of existing chemical pesticides and the instability of naked dsRNA as a pesticide. This invention aims to provide a highly stable, targeted, and environmentally friendly RNA biopesticide and its preparation method. This pesticide can effectively silence the key HcAmy2 gene of the fall webworm, disrupting its multi-host adaptability, thereby achieving highly efficient control.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] According to the first aspect of this disclosure, an RNA biopesticide targeting the HcAmy2 gene of the fall webworm is proposed. The active ingredient of the biopesticide is a double-stranded RNA targeting the α-amylase gene of the fall webworm, namely dsHcAmy2, and is encapsulated by chitosan to form a chitosan / dsRNA nanocomposite. The degree of deacetylation of the chitosan is ≥95%, and the viscosity is 100-200 mPa·s.

[0010] Furthermore, the chitosan is dissolved in 0.1 M sodium acetate buffer at pH 4.5, and the final concentration in the nanocomposite is 0.005%-0.02% (w / v); the dsHcAmy2 is present in a solution containing 2.5 mol / L Na2SO4 before the formation of the composite.

[0011] According to the second aspect of this disclosure, a method for preparing an RNA biopesticide targeting the HcAmy2 gene of the fall webworm is also provided, comprising the following steps:

[0012] S1. Based on the coding sequence of the HcAmy2 gene of the fall webworm, specific primers with the T7 promoter were designed and synthesized; PCR amplification was performed using the HcAmy2 cloning vector as a template to obtain the dsHcAmy2 fragment; the purified PCR product was sequenced for verification.

[0013] S2. Using an in vitro transcription kit, and with the PCR product verified as correct in step S1 as a template, synthesize dsHcAmy2; and synthesize irrelevant sequence double-stranded RNA, i.e., dsGFP, as a negative control; dissolve the synthesized dsHcAmy2 in nuclease-free water to obtain dsHcAmy2 solution for later use.

[0014] S3. Dissolve chitosan with a degree of deacetylation ≥95% and a viscosity of 100-200 mPa·s in 0.1 M sodium acetate buffer solution at pH 4.5 to prepare chitosan sodium acetate solutions with mass-volume concentrations of 0.02%, 0.01%, and 0.005%.

[0015] S4. Dilute the dsHcAmy2 solution obtained in step S2 with DEPC water to the target concentration, and add Na2SO4 to make the final concentration 2.5 mol / L to obtain the pretreated dsHcAmy2 solution.

[0016] S5. Mix the chitosan sodium acetate solution prepared in step S3 with the pretreated dsHcAmy2 solution obtained in step S4 in equal volumes to obtain a mixture; incubate the mixture at 55°C for 1 minute, then vortex for 30 seconds, and then let it stand at room temperature for 30 minutes to form a chitosan / dsRNA nanocomposite.

[0017] S6. The encapsulation effect of the dsHcAmy2 was verified by agarose gel electrophoresis; the stability of the complex at different time points was evaluated using midgut fluid from fall webworm larvae; and the morphology of the chitosan / dsRNA nanocomposite was characterized by transmission electron microscopy and scanning electron microscopy.

[0018] Furthermore, in step S1, the primers used to amplify the dsHcAmy2 fragment are specific primers with a T7 promoter sequence.

[0019] Further, in step S4, the pretreatment of the dsHcAmy2 solution includes adding Na2SO4 to the dsHcAmy2 solution to achieve a final concentration of 2.5 mol / L.

[0020] Furthermore, in step S5, the composite conditions of chitosan and dsHcAmy2 are: incubation at 55°C for 1 minute, vortexing for 30 seconds, and standing at room temperature for 30 minutes.

[0021] Further, in step S6, the stability assessment includes: co-incubating the chitosan / dsRNA nanocomposite with the midgut fluid of fifth-instar larvae of the fall webworm at 25°C, taking samples at 0.5, 1, 2, 4, and 6 hours respectively, detecting the integrity of dsHcAmy2 by agarose gel electrophoresis, and using unencapsulated naked dsHcAmy2 as a control.

[0022] Furthermore, in step S6, the morphology, particle size, and dispersibility of the chitosan / dsRNA nanocomposite are characterized using transmission electron microscopy and scanning electron microscopy.

[0023] According to the third aspect of this disclosure, a method for verifying the biosafety of an RNA biopesticide targeting the HcAmy2 gene of the fall webworm is also proposed, including the following verification methods:

[0024] a. Impact assessment on non-target lepidopteran insects: Healthy, uniformly sized second-instar larvae of the gypsy moth were selected and fed with artificial feed coated with chitosan / dsRNA nanocomposite for 2 days. They were then transferred to normal feed for 4 days. The mortality rate and weight changes of the larvae were recorded and compared with the distilled water control and the CS-dsGFP negative control group.

[0025] b. Impact assessment on non-target natural enemy insects: Using the dominant natural enemy insect, the stink bug, as the test subject, after 24 hours of treatment with chitosan / dsRNA nanocomposite, its predation rate under different prey densities was measured. The Holling type II functional response equation, Na=aNT / (1+aThN), was fitted to calculate the instantaneous attack rate a, treatment time Th, and search efficiency S=a / (1+aThN) to assess its impact on predation ability.

[0026] c. Toxicity assessment of non-target insect cells: The cytotoxicity of chitosan / dsRNA nanocomplex to fall armyworm ovarian cells was determined using the CCK-8 cell viability assay, with distilled water and CS-dsGFP as controls. Cell viability was detected after 48 hours of treatment.

[0027] According to the fourth aspect of this disclosure, the application of RNA biopesticides as described in the first aspect of this disclosure as plant-derived pesticide synergists is also proposed, by combining the chitosan / dsRNA nanocomposite with coumarin-based plant-derived insecticides for synergistic control of fall webworm larvae.

[0028] Compared with existing technologies, this invention provides an RNA biopesticide targeting the HcAmy2 gene of the fall webworm and its preparation method. This biopesticide targets the key digestive enzyme gene HcAmy2 in the fall webworm, specifically inhibiting its expression via RNAi. This significantly reduces the weight of larvae after feeding on different host plants and increases their mortality rate, effectively disrupting their multi-host adaptability. By encapsulating dsRNA with chitosan nanocarriers, the resulting complex effectively resists degradation by nucleases in the insect midgut fluid and remains stable for at least 6 hours in a simulated midgut environment, greatly extending the field persistence of the dsRNA. Chitosan is a natural biodegradable material, and biosafety tests show that this formulation has no significant toxicity to non-target insects (gypsy moth), important natural enemies (insect bugs), and insect cells (sf9 cells), indicating low ecological risk. This RNA biopesticide exhibits a significant synergistic effect when used in combination with plant-derived insecticides such as coumarin. The overall preparation method is mild, with clear steps, good reproducibility, and easy to scale up for production. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0030] Figure 1 Gel retardation electrophoresis diagram and midgut fluid stability detection diagram of the chitosan / dsRNA nanocomposite provided in the embodiments of the present invention;

[0031] Figure 2 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) characterization images of chitosan / dsRNA nanocomposites are provided for embodiments of the present invention;

[0032] Figure 3 The silencing efficiency evaluation results of the chitosan / dsRNA nanocomposite provided in the embodiments of the present invention;

[0033] Figure 4 The safety assessment results of the chitosan / dsRNA nanocomposite on non-target organisms (gypsy moth, volcano bug, Sf9 cells) provided in the embodiments of the present invention.

[0034] Figure 5 The figure shows the effect of chitosan / dsRNA nanocomposite treatment provided in this embodiment of the invention on the weight and mortality of American white moth larvae after feeding on three host plants.

[0035] Figure 6 The chitosan / dsRNA nanocomposite combined with coumarin provided in this embodiment of the invention exhibits a synergistic effect on the larvae of the fall webworm. Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0037] Example 1: Synthesis of target dsHcAmy2 and preparation of chitosan / dsRNA nanocomposite

[0038] The purpose of this embodiment is to prepare an RNA biopesticide (CS-dsHcAmy2 nanocomposite) targeting the HcAmy2 gene of the fall webworm, and to verify and characterize the encapsulation effect, stability and morphology of the composite.

[0039] 1. Materials and reagents:

[0040] American white moth HcAmy2 gene cloning vector, T7 RiboMAX™ Express RNAi system, nuclease-free water, DEPC water, chitosan (degree of deacetylation ≥95%, viscosity 100-200 mPa.s), sodium acetate, glacial acetic acid, sodium sulfate (Na2SO4).

[0041] 2. Preparation and Validation of the dsHcAmy2 Template

[0042] Primers containing the T7 promoter were designed based on the coding sequence of the HcAmy2 gene of the American white moth. Using the HcAmy2 cloning vector as a template, PCR amplification was performed using the above primers. The PCR product was recovered and purified by agarose gel electrophoresis to obtain a DNA fragment of approximately 494 bp. The purified PCR product was sequenced to verify that its sequence was completely consistent with the corresponding fragment of the target HcAmy2 gene.

[0043] 3. In vitro synthesis of dsHcAmy2

[0044] Sequencing verification confirmed that the T7 RiboMAX™ Express RNAi system was used to perform in vitro transcription using the verified PCR product as a template to synthesize double-stranded RNA (dsHcAmy2).

[0045] Using a plasmid containing the green fluorescent protein (GFP) gene sequence as a template, an unrelated double-stranded RNA (dsGFP) was synthesized as a negative control for subsequent experiments.

[0046] The synthesized dsHcAmy2 and dsGFP were dissolved in an appropriate amount of nuclease-free water for later use.

[0047] 4. Preparation of chitosan solution

[0048] Chitosan powder (degree of deacetylation ≥ 95%) was dissolved in 0.1 M sodium acetate buffer solution at pH 4.5 and magnetically stirred until completely dissolved.

[0049] Prepare chitosan sodium acetate solutions with w / v concentrations of 0.02%, 0.01%, and 0.005%, respectively, filter them through a 0.22 μm microporous membrane for sterilization, and store them at 4°C for later use.

[0050] 5. Preprocessing of dsHcAmy2

[0051] The dsHcAmy2 solution was diluted with DEPC water to 60 μg / 100 μL. Solid Na2SO4 was added to the diluted dsHcAmy2 solution and vortexed to dissolve it completely, so that the final concentration of Na2SO4 was 2.5 mol / L.

[0052] 6. Preparation of chitosan / dsRNA nanocomposites

[0053] Take equal volumes of chitosan solution and pretreated dsHcAmy2 solution and mix them in a sterile centrifuge tube;

[0054] The mixture was incubated in a 55°C metal bath for 1 minute, and then the centrifuge tube was quickly placed on a vortex mixer and vortexed for 30 seconds. The centrifuge tube was then allowed to stand at room temperature for 30 minutes to obtain the chitosan / dsRNA nanocomposite (CS / dsHcAmy2). A negative control complex (CS / dsGFP) with dsGFP as the core was prepared using the same method.

[0055] 7. Verification and characterization of the complex

[0056] Gel retardation experiment: 10 μL of the prepared CS / dsHcAmy2 complex, CS / dsGFP complex, and an equal amount of free dsHcAmy2 were subjected to 1% agarose gel electrophoresis (100 V, 30 min). The results are as follows: Figure 1 As shown in Figure A, compared to the normal migration of free dsRNA in the gel, the dsRNA in the CS / dsHcAmy2 and CS / dsGFP complexes was completely blocked within the sample wells, indicating that chitosan successfully encapsulated the dsRNA, forming a nanocomposite. Comparing the encapsulation effects at different chitosan concentrations (0.02%, 0.01%, and 0.005%), the 0.02% concentration showed the most complete encapsulation.

[0057] Midgut fluid stability test: Midgut fluid was extracted from fifth-instar larvae of the fall webworm. A CS / dsHcAmy2 complex (0.02% CS group) was mixed with the midgut fluid at a specific ratio and incubated at 25°C for 0, 0.5, 1, 2, 4, and 6 hours, respectively. Samples were taken at each time point for agarose gel electrophoresis analysis. The results are as follows: Figure 1 As shown in Figure B, the CS / dsHcAmy2 complex maintained the integrity of dsHcAmy2 at all time points, while naked dsHcAmy2 showed obvious degradation bands after 0.5 hours of incubation. This indicates that chitosan encapsulation can effectively protect dsRNA from degradation by nucleases in the midgut fluid, significantly improving its stability.

[0058] Morphological characteristics:

[0059] ① Scanning electron microscopy (SEM) observation: A small amount of CS / dsHcAmy2 composite was dropped onto a silicon wafer, allowed to dry naturally, and then sputter-coated with gold. Under SEM observation (accelerating voltage 5 kV), the composite appeared as irregular, porous, sponge-like or network-like aggregates. Figure 2 A) It has a large specific surface area;

[0060] ② Transmission electron microscopy (TEM) observation: The complex was dropped onto a copper grid and observed after negative staining. TEM image ( Figure 2 B) shows that the complex exhibits a distinct core-shell structure, with a dense black core (corresponding to dsHcAmy2) at the center and surrounded by a light gray, translucent chitosan layer, with a relatively uniform particle size distribution.

[0061] Example 2: Indoor silencing efficiency determination of fall webworm larvae

[0062] The purpose of this embodiment is to verify the silencing effect of the CS-dsHcAmy2 nanocomposite on the HcAmy2 gene in the larvae of the fall webworm.

[0063] 1. Feed processing

[0064] Take CS-dsHcAmy2 complex, CS-dsGFP complex and distilled water (blank control) respectively, and spread them evenly on the surface of artificial feed cooled to 45℃ at a ratio of 1g artificial feed: 100μL sample. Gently spread them evenly with a sterile glass rod and place them in a clean bench to air dry (about 30min) to avoid residual liquid on the feed surface.

[0065] 2. Larval feeding and sample collection

[0066] The treated artificial feed was placed into sterile rearing boxes, with 20 third-instar early larvae of the fall webworm in each box. Each treatment group had 5 replicates. The rearing conditions were 25±1℃, 60%-70% relative humidity, and 16L:8D photoperiod. After 48 hours of continuous feeding, 10 larvae from each group were randomly selected, and the midgut tissue was dissected, quickly frozen in liquid nitrogen, and stored at -80℃ for later use.

[0067] 3. RNA extraction and cDNA synthesis

[0068] Total RNA was extracted from the midgut tissue of larvae according to the RNA extraction kit instructions. RNA purity (A260 / A280 ratio between 1.8 and 2.0) and concentration were detected using Nanodrop, and RNA integrity was verified by 1% agarose gel electrophoresis. Using the total RNA as a template, cDNA was synthesized using a reverse transcription kit. The reaction conditions were: reverse transcription at 42℃ for 30 min, inactivation at 85℃ for 5 min, and storage at -20℃.

[0069] 4. qPCR detection

[0070] Using cDNA as a template, qPCR detection was performed using specific primers for the HcAmy2 gene, with the β-actin gene of the fall webworm (Moth simonii) as an internal reference gene. Two... - The relative expression level of the HcAmy2 gene was calculated using the ΔΔCt method.

[0071] 5. Experimental Results

[0072] qPCR results showed that, compared with the distilled water blank control group and the CS-dsGFP negative control group, the relative expression level of HcAmy2 gene in the midgut of fall webworm larvae treated with the CS-dsHcAmy2 complex was significantly decreased, with an average silencing efficiency of 47.39% (e.g., ...). Figure 3 As shown in the figure, this indicates that the CS-dsHcAmy2 complex can effectively enter the larvae, triggering an RNA interference response in the target gene and silencing the target gene.

[0073] Example 3: Safety assessment of non-target organisms

[0074] The purpose of this embodiment is to evaluate the safety of the CS-dsHcAmy2 nanocomposite to non-target insects, natural enemy insects, and insect cells.

[0075] 1. Toxicity assessment on non-target insect cells (Sf9 cells)

[0076] Cell culture: Sf9 cells were seeded in RPMI-1640 medium containing 10% fetal bovine serum and cultured at 27°C in a 5% CO2 incubator until the logarithmic growth phase. After trypsin digestion, the cell concentration was adjusted to 1×10⁶ cells / year. 5 Cells / mL were seeded into 96-well cell culture plates, 100 μL per well, and cultured for 24 h.

[0077] Sample preparation: Dilute the CS-dsHcAmy2 complex with culture medium to a final concentration equivalent to that in vivo, add 10 μL to each well, and gently shake to mix; set up a distilled water control group, a CS-dsGFP control group, and a blank culture medium control group, with 6 replicates in each group, and continue to incubate for 48 h;

[0078] Cell viability assay: Following the instructions of the CCK-8 kit, add 10 μL of CCK-8 solution to each well and continue culturing for 4 h. Then, measure the absorbance (OD value) of each well at a wavelength of 450 nm using a microplate reader and calculate the cell viability (cell viability = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%).

[0079] Experimental results: The viability of Sf9 cells in the CS-dsHcAmy2 treatment group was not significantly different from that in the distilled water control group and the CS-dsGFP control group (P>0.05), indicating that this complex has no significant toxicity to non-target insect cells (e.g., Figure 4 (As shown in A).

[0080] 2. Impact assessment on non-target lepidopteran insects (gypsy moth)

[0081] Feed treatment: Following the method in Example 2, the CS-dsHcAmy2 complex, the CS-dsGFP complex, and distilled water were respectively coated onto the surface of the artificial feed for gypsy moths and then dried for later use.

[0082] Healthy, uniform second-instar larvae of the gypsy moth were selected, with 30 larvae per treatment group and 3 replicates. Each treatment group was fed the corresponding artificial feed for 2 days, and then transferred to the untreated normal artificial feed for another 4 days. The survival status of the larvae was recorded daily. After the feeding period, the weight of each larva was measured, and the mortality rate and average weight were calculated.

[0083] Experimental results: After the rearing period, the mortality rate of gypsy moth larvae in the CS-dsHcAmy2 treatment group was not significantly different from that in the distilled water control group and the CS-dsGFP control group (P>0.05); the average body weight was also not significantly different from that in the control group (P>0.05). Figure 4 (As shown in B and C).

[0084] 3. Impact assessment on natural enemy insects (insect bugs)

[0085] Treatment of the stink bug: Adult stink bugs were placed in a culture dish containing a solution of CS-dsHcAmy2 complex (concentration as in Example 1), and their body surface was in contact with the solution for 10 seconds. Then, they were transferred to a sterile culture dish, and the residual liquid on the body surface was blotted with filter paper. The dish was then placed in an incubator at 25°C for 24 hours. A distilled water treatment group and a CS-dsGFP treatment group were set up as controls.

[0086] Predation response determination: After treatment, adult stink bugs were starved for 24 hours and then placed in petri dishes (9 cm in diameter) containing 5, 10, 15, 20, and 25 third-instar larvae of the fall webworm, respectively. Five replicates were set for each prey density. After being reared at 25°C for 24 hours, the predation rate of the stink bugs was recorded. The Holling type II functional response equation (Na=aNT / (1+aThN)) was used for fitting to calculate the instantaneous attack rate (a), treatment time (Th), and search efficiency (S=a / (1+aThN)).

[0087] Experimental Results: Holling type II equation fitting results showed that the instantaneous attack rate, treatment time, and searching efficiency of the CS-dsHcAmy2 treated group were not significantly different from those of the distilled water control group and the CS-dsGFP control group (P>0.05). Figure 4 (As shown in DF) This indicates that the preparation does not affect the predation ability of the bug.

[0088] Example 4: Field application potential and synergistic effect trial

[0089] The purpose of this embodiment is to verify the control effect of CS-dsHcAmy2 nanocomposite on the larvae of the fall webworm that feed on different host plants, and its synergistic effect with plant-derived pesticides.

[0090] 1. Control efficacy against fall webworm larvae on different host plants

[0091] Leaf treatment: Select fresh leaves of Manchurian ash, birch and linden of uniform size, and spray them evenly with CS-dsHcAmy2 complex, CS-dsGFP complex and distilled water respectively. The amount of spraying is 1 mL per leaf. After air drying, place them in a sterile petri dish with a diameter of 15 cm (lined with moist filter paper to maintain humidity).

[0092] Larval feeding and observation: 10 third-instar larvae of the fall webworm were placed in each culture dish. Five replicates were set up for each treatment group. The survival of the larvae was observed and recorded every day. After the feeding was completed, the weight of each larva was weighed and the weight inhibition rate and mortality rate were calculated (weight inhibition rate = (average weight of control group - average weight of treatment group) / average weight of control group × 100%).

[0093] Experimental results: The CS-dsHcAmy2 treatment group showed significant control effects on different host plants of the American white moth, significantly reducing the larval weight (inhibition rate 17.89%-47.97%) and increasing the mortality rate (2.38-5.20 times) on all three host plants. Figure 5 (As shown).

[0094] 2. Synergistic effect with coumarin

[0095] Group setup: Four treatment groups were set up: ① CS-dsHcAmy2 treatment group alone; ② Coumarin treatment group alone; ③ CS-dsHcAmy2 + coumarin combined treatment group; ④ Distilled water blank control group.

[0096] Treatment methods: ① Group: Larvae were fed artificial feed containing CS-dsHcAmy2 complex for 2 days according to the method in Example 2; ② Group: Coumarin was added to the artificial feed to a final concentration of 2 mg / g and fed directly to the larvae; ③ Group: Larvae were first fed artificial feed containing CS-dsHcAmy2 complex for 2 days, and then fed artificial feed containing 2 mg / g coumarin for 24 hours; ④ Group: Larvae were fed untreated artificial feed.

[0097] Effect detection: Five replicates were set for each treatment group, with 20 larvae in each replicate. After the feeding ended, the larval mortality was recorded. The co-toxicity coefficient (CTC) was calculated using the Colby method to judge the synergistic effect (synergistic effect when CTC > 120, additive effect when 80 < CTC ≤ 120, and antagonistic effect when CTC ≤ 80). Meanwhile, χ² test was conducted for verification.

[0098] Experimental results: The detection results after 24 h showed that the larval mortality of the combined treatment group of CS-dsHcAmy2 and coumarin was significantly higher than the expected additive mortality of the two treated alone. The χ² test indicated that the two had a synergistic effect (as Figure 6 shown), suggesting that CS-dsHcAmy2 and coumarin had a significant synergistic effect.

[0099] In summary, the present invention successfully prepared a stable and highly efficient RNA biopesticide with chitosan as the carrier and targeting the HcAmy2 gene of Hyphantria cunea. This preparation can effectively silence the target gene, significantly inhibit the growth and development of pests, is safe for non-target organisms, and can synergistically enhance the effect with plant-derived pesticides, showing good prospects for development and application.

[0100] Only some exemplary embodiments of the present invention have been described by way of illustration. Undoubtedly, for those of ordinary skill in the art, the described embodiments can be modified in various different ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the protection scope of the claims of the present invention.

Claims

1. An RNA biopesticide targeting the HcAmy2 gene of the fall webworm, characterized in that, The active ingredient of the biopesticide is a double-stranded RNA targeting the α-amylase gene of the fall webworm, namely dsHcAmy2, and is encapsulated by chitosan to form a chitosan / dsRNA nanocomposite; the degree of deacetylation of the chitosan is ≥95%, and the viscosity is 100-200 mPa.s.

2. The RNA biopesticide targeting the HcAmy2 gene of the fall webworm according to claim 1, characterized in that, The chitosan was dissolved in 0.1 M sodium acetate buffer at pH 4.5, and the final concentration in the nanocomposite was 0.005%-0.02% (w / v); the dsHcAmy2 was present in a solution containing 2.5 mol / L Na2SO4 before the formation of the composite.

3. A method for preparing an RNA biopesticide targeting the HcAmy2 gene of the fall webworm as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Based on the coding sequence of the HcAmy2 gene of the fall webworm, specific primers with the T7 promoter were designed and synthesized. PCR amplification was performed using the HcAmy2 cloning vector as a template to obtain the dsHcAmy2 fragment; the purified PCR product was then sequenced for verification. S2. Using an in vitro transcription kit, and with the PCR product verified as correct in step S1 as a template, synthesize dsHcAmy2; and synthesize irrelevant sequence double-stranded RNA, i.e., dsGFP, as a negative control; dissolve the synthesized dsHcAmy2 in nuclease-free water to obtain dsHcAmy2 solution for later use. S3. Dissolve chitosan with a degree of deacetylation ≥95% and a viscosity of 100-200 mPa·s in 0.1 M sodium acetate buffer solution at pH 4.5 to prepare chitosan sodium acetate solutions with mass-volume concentrations of 0.02%, 0.01%, and 0.005%. S4. Dilute the dsHcAmy2 solution obtained in step S2 with DEPC water to the target concentration, and add Na2SO4 to make the final concentration 2.5 mol / L to obtain the pretreated dsHcAmy2 solution. S5. Mix the chitosan sodium acetate solution prepared in step S3 with the pretreated dsHcAmy2 solution obtained in step S4 in equal volumes to obtain a mixture; incubate the mixture at 55°C for 1 minute, then vortex for 30 seconds, and then let it stand at room temperature for 30 minutes to form a chitosan / dsRNA nanocomposite. S6. The encapsulation effect of the dsHcAmy2 was verified by agarose gel electrophoresis; the stability of the complex at different time points was evaluated using midgut fluid from fall webworm larvae; and the morphology of the chitosan / dsRNA nanocomposite was characterized by transmission electron microscopy and scanning electron microscopy.

4. The method for preparing an RNA biopesticide targeting the HcAmy2 gene of the fall webworm according to claim 3, characterized in that, In step S1, the primers used to amplify the dsHcAmy2 fragment are specific primers with the T7 promoter sequence.

5. The method for preparing an RNA biopesticide targeting the HcAmy2 gene of the fall webworm according to claim 3, characterized in that, In step S4, the pretreatment of the dsHcAmy2 solution includes adding Na2SO4 to the dsHcAmy2 solution to achieve a final concentration of 2.5 mol / L.

6. The method for preparing an RNA biopesticide targeting the HcAmy2 gene of the fall webworm according to claim 3, characterized in that, In step S5, the composite conditions of chitosan and dsHcAmy2 are as follows: incubation at 55°C for 1 minute, vortexing for 30 seconds, and standing at room temperature for 30 minutes.

7. The method for preparing an RNA biopesticide targeting the HcAmy2 gene of the fall webworm according to claim 3, characterized in that, In step S6, the stability assessment includes: co-incubating the chitosan / dsRNA nanocomposite with the midgut fluid of fifth-instar larvae of the fall webworm at 25°C, taking samples at 0.5, 1, 2, 4, and 6 hours respectively, detecting the integrity of dsHcAmy2 by agarose gel electrophoresis, and using unencapsulated naked dsHcAmy2 as a control.

8. The method for preparing an RNA biopesticide targeting the HcAmy2 gene of the fall webworm according to claim 3, characterized in that, In step S6, the morphology, particle size and dispersibility of the chitosan / dsRNA nanocomposite are characterized using transmission electron microscopy and scanning electron microscopy.

9. The biosafety of the RNA biopesticide targeting the HcAmy2 gene of the fall webworm according to claim 1 or 2, characterized in that, The following verification methods are included: a. Impact assessment on non-target lepidopteran insects: Healthy, uniformly sized second-instar larvae of the gypsy moth were selected and fed with artificial feed coated with chitosan / dsRNA nanocomposite for 2 days. They were then transferred to normal feed for 4 days. The mortality rate and weight changes of the larvae were recorded and compared with the distilled water control and the CS-dsGFP negative control group. b. Impact assessment on non-target natural enemy insects: Using the dominant natural enemy insect, the stink bug, as the test subject, after 24 hours of treatment with chitosan / dsRNA nanocomposite, its predation rate under different prey densities was measured. The Holling type II functional response equation, Na=aNT / (1+aThN), was fitted to calculate the instantaneous attack rate a, treatment time Th, and search efficiency S=a / (1+aThN) to assess its impact on predation ability. c. Toxicity assessment of non-target insect cells: The cytotoxicity of chitosan / dsRNA nanocomplex to fall armyworm ovarian cells was determined using the CCK-8 cell viability assay, with distilled water and CS-dsGFP as controls. Cell viability was detected after 48 hours of treatment.

10. The application of an RNA biopesticide as described in claim 1 or 2 as a plant-derived pesticide synergist, characterized in that: The chitosan / dsRNA nanocomposite was used in combination with a coumarin-based plant-derived insecticide for synergistic control of fall webworm larvae.

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