Small interfering RNA (Ribonucleic Acid) nano biological agent for preventing and treating trunk borers and preparation method of small interfering RNA nano biological agent

Abstract

The invention discloses a small interfering RNA nano biological agent for preventing and treating trunk borers and a preparation method of the small interfering RNA nano biological agent, and belongs to the technical field of agricultural pest prevention and treatment. The preparation method comprises the following steps: adsorbing small interfering RNA (siRNA) on the surface of gold nanoparticles under the action of charges, then embedding into copper-based organic metal framework nanoparticles, then acting with chitosan to form a chitosan nano-film on the surface, and finally emulsifying with polyether modified heptamethyltrisiloxane, so as to obtain the copper-based organic metal framework nano-particle / chitosan nano-film / polyether modified heptamethyltrisiloxane / chitosan nano-film composite material. The precise intelligent small interfering RNA nano biological agent with high permeability, high adsorption performance and film-forming property is prepared and can be used for effectively preventing and treating trunk borers.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural pest control, and in particular to a small interfering RNA nanobiological agent for controlling stem-boring pests and a preparation method thereof. Background Art

[0002] Trunk-boring insects are insects that bore into the trunk or branches of trees at a certain stage of their development. There are approximately 119,000 trunk-boring pest species worldwide, with Monochamus alternatus being a prime example and a major forestry quarantine pest. Its larvae weaken the phloem and xylem of pine trees, disrupting water and nutrient transport and causing wood dieback. Adults, while feeding and replenishing their nutrition, gnaw on young branches, carrying pine wood nematodes. These nematodes invade pine trees, infecting them with wounds and causing weakened growth, ultimately leading to widespread mortality and severe economic and ecological losses. Currently, the main methods for controlling trunk-boring pests include chemical control, attractants, and biological control. Chemical control currently relies primarily on proactive preventive measures such as trunk injection. However, the effectiveness of chemical trunk injection is affected by numerous environmental factors and can cause additional damage to the tree.

[0003] RNA interference (RNAi) refers to the highly conserved phenomenon of efficient and specific degradation of homologous messenger RNA induced by double-stranded RNA (dsRNA) during evolution. It can induce a loss-of-function phenotype by inhibiting gene expression at the pre-transcriptional or post-transcriptional level. Nucleic acid pesticides are a class of polynucleotides that can specifically bind to mRNA transcribed from specific genes in target organisms. They silence the expression of corresponding genes through the naturally occurring RNAi system in the target organisms, thereby interfering with the normal growth of the target organisms and their harm to the host plant, ultimately achieving the purpose of protecting plants. Nucleic acid pesticides have the advantages of strong specificity, rapid effect, no residue, and minimal impact on non-target organisms. They are hailed as the third revolution in the history of pesticides and are a research hotspot in the creation of new green pesticides. However, their promotion and use in farmland have the disadvantages of low gene interference efficiency, low prevention efficacy, and short duration of effect, which seriously restrict the field application of RNA preparations.

[0004] Nanotechnology can improve the mode of action of pesticides on their targets, their entry pathways into insects and pathogens, and their efficiency, thereby enhancing their biological activity and toxicity. The encapsulation and adsorption of drugs in nanocarriers achieves efficient drug delivery, enabling controlled and sustained release and prolonging their duration of action. There are reports on the use of nanocarriers to deliver nucleic acid pesticides. For example, Zheng et al. reported a mixture containing a surfactant, a cationic nanocarrier, and dsRNA that rapidly penetrates the aphid body wall and enters the hemocoel, effectively improving dsRNA delivery efficiency. Yang et al. reported that nanocarrier-delivered miRNAs were efficiently absorbed by Arabidopsis thaliana and maize. However, stem-boring pests spend most of their life cycle hidden, exposed only to the external environment during their brief adult stage. This makes their control more difficult than other pest species. Furthermore, drug entry and delivery within forest trunks are even more challenging, making the non-invasive and efficient control of stem-boring pests a major challenge. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention aims to provide a small interfering RNA nanoparticle biopharmaceutical for controlling stem-boring pests and its preparation method. In this method, small interfering RNA (siRNA) is adsorbed on the surface of gold nanoparticles through charge interaction, then embedded into copper-based organometallic framework nanoparticles. This then interacts with chitosan to form a chitosan nanofilm, which is then emulsified with polyether-modified heptamethyltrisiloxane. The resulting nanoparticle is a precise, intelligent small interfering RNA biopharmaceutical with high permeability and adsorption properties, which can be used to effectively control stem-boring pests.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, a small interfering RNA nanopharmaceutical is provided, which is prepared by the following method:

[0008] (1) mixing a polyethyleneimine solution and polyethylene glycol-stabilized gold nanoparticles, adding small interfering RNA, and then adding the polyethyleneimine solution to obtain a gold nanoparticle-loaded small interfering RNA nanopesticide;

[0009] (2) dissolving copper nitrate and polyvinyl pyrrolidone in methanol to obtain a first solution, and dissolving 1,3,5-benzenetricarboxylic acid in methanol to obtain a second solution; adding the small interfering RNA nanopesticide loaded with gold nanoparticles to the first solution, mixing well, and then adding the second solution, standing, centrifuging, collecting the precipitate, washing, and drying to prepare the small interfering RNA co-loaded with gold nanoparticles / organic metal framework;

[0010] (3) The small interfering RNA co-loaded by the gold nanoparticles / organic metal framework is dispersed in deionized water, chitosan solution is added, and the precipitate is collected by centrifugation after the reaction. The precipitate is redispersed in deionized water, polyether-modified heptamethyltrisiloxane is added, and ultrasonic emulsification is performed to prepare the small interfering RNA nanobiological agent.

[0011] Preferably, in step (1), the concentration of the polyethyleneimine solution is 0.1-2 mg / mL. The polyethyleneimine acts as a positive electrolyte and can adsorb small interfering RNA onto the surface of gold nanoparticles.

[0012] Preferably, in step (1), the polyethylene glycol-stabilized gold nanoparticles are prepared by the following method:

[0013] The polyethylene glycol solution was added dropwise to the gold nanoparticles, stirred for 2-3 hours, centrifuged, and washed to prepare polyethylene glycol-stabilized gold nanoparticles.

[0014] The above treatment of gold nanoparticles with polyethylene glycol can prevent the aggregation of gold nanoparticles and enhance their stability in solution.

[0015] Preferably, in step (1), the small interfering RNA is a small interfering RNA targeting growth and development genes, reproductive genes and / or olfactory genes of stem-boring pests, thereby preventing and controlling stem-boring pests by interfering with the expression of key genes of stem-boring pests.

[0016] More preferably, the small interfering RNA targets the Monochamus alternatus chitin deacetylase gene (MaCDA2b, GenBank: KY914480.1).

[0017] Preferably, in step (2), the concentration of copper nitrate in the first solution is 0.005 g / mL-0.015 g / mL, and the concentration of polyvinyl pyrrolidone is 0.001 g / mL-0.003 g / mL; the concentration of 1,3,5-benzenetricarboxylic acid in the second solution is 0.001 g / mL-0.01 g / mL; and the volume ratio of the first solution to the second solution is 1:1.

[0018] Preferably, in step (3), the mass ratio of the small interfering RNA co-loaded by the gold nanoparticles / organic metal framework to chitosan is 1:10-10:1; the mass ratio of the small interfering RNA co-loaded by the gold nanoparticles / organic metal framework to polyether-modified heptamethyltrisiloxane is 1:100-1000:1.

[0019] Preferably, in step (3), the ultrasonic reaction time is 10-120 min.

[0020] The second aspect of the present invention provides the use of the above-mentioned small interfering RNA nanobiological agent in the following (1) or (2):

[0021] (1) Prevent and control stem-boring pests;

[0022] (2) Preparation of products for controlling stem borers.

[0023] In the above application, the trunk-boring pests include but are not limited to: longhorn beetles, wood borers, jellyfish, bark beetles and weevils.

[0024] A third aspect of the present invention provides a method for controlling stem borers, comprising the following steps:

[0025] Before or when the trunk-boring pests attack the tree, the above-mentioned small interfering RNA nano-biological agent is sprayed on the surface of the tree trunk.

[0026] Beneficial effects of the present invention:

[0027] (1) The present invention adsorbs small interfering RNA on the surface of gold nanoparticles through charge action, then embeds it into copper-based organic metal framework nanoparticles, and then reacts with chitosan to form a chitosan nanofilm on the surface. Finally, it is emulsified with polyether-modified heptamethyltrisiloxane to prepare a precise intelligent small interfering RNA nanobiological agent with high permeability and high adsorption performance.

[0028] (2) The small interfering RNA nanobiological agent prepared by the present invention has both pH and enzyme dual-responsive controlled release of small interfering RNA, protects small interfering RNA and prolongs the duration of RNAi effect, thereby improving the enrichment ability in target genes.

[0029] (3) The small interfering RNA nanobiological agent prepared by the present invention contains copper-based organometallic skeleton nanoparticles that are biodegradable and release copper ions, which have both disease resistance and bactericidal effects and can synergistically control pine wood nematode disease.

[0030] (4) The small interfering RNA nanobiological agent prepared by the present invention does not require drilling, can be accurately applied, reduce the amount and increase the efficiency, and can silence the key target genes of stem-boring pests, reducing the pests' survival ability or even killing them.

[0031] (5) The small interfering RNA nanopharmaceuticals prepared by the present invention can also encapsulate a variety of small molecule pesticides to achieve co-carrying, co-delivery, high efficiency, and synergistic control of pests and diseases, and are suitable for individual and combined prevention and control in various scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Transmission electron microscopy (A) and scanning electron microscopy (B) images of the small interfering RNA co-loaded by the gold nanoparticles / organic metal framework prepared in Example 1 of the present invention.

[0033] Figure 2: Element distribution diagram of small interfering RNA co-loaded by gold nanoparticles / organic metal framework prepared in Example 1 of the present invention.

[0034] Figure 3 : The sustained release curves of the small interfering RNA nanobiological agent prepared in Example 1 of the present invention at temperature conditions of 15°C (A), 25°C (B), and 35°C (C) and different pH conditions (pH = 6.0, 7.4, and 8.2).

[0035] Figure 4 : Delivery of the small interfering RNA nanobiological agent prepared in Example 1 of the present invention in the larvae of the pine beetle; in the figure, A is a fluorescence image of the small interfering RNA nanobiological agent; B is a fluorescence distribution image of the pine beetle larvae after treatment with the small interfering RNA nanobiological agent; C is the fluorescence distribution and magnified image of the stomata of the pine beetle larvae; D is a bright field HE image and fluorescence distribution image of the cross section of the midgut of the pine beetle larvae.

[0036] Figure 5 : Fluorescence intensity distribution diagram of the cross section of pine trunk treated with small interfering RNA nanopharmaceutical prepared in Example 1 at different concentrations and blank control diagram; in the figure, AD represents the treatment concentrations of 20, 40, 60, and 80 μg / 10 mL, respectively.

[0037] Figure 6 : Fluorescence intensity distribution diagram of the cross section of pine trunk treated with the small interfering RNA nanobiological agent prepared in Example 1 at different time points and a blank control diagram; in the figure, AD represents the time points of 1 h, 3 h, 5 h and 7 h, respectively.

[0038] Figure 7 : The relative expression level of chitin deacetylase gene after contact killing and stomach poisoning of Monochamus alternatus larvae by the small interfering RNA nanobiological agent prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0040] As mentioned above, stem-boring pests are difficult to control. Monochamus alternatus is a typical stem-boring pest. Adults lay their eggs in the bark until they emerge from the openings. Their sheltered habitat creates a natural barrier to control. Existing RNA interference drugs struggle to effectively control stem-boring pests.

[0041] In light of this, the present invention has developed a small interfering RNA (siRNA) nanoparticle biopharmaceutical for controlling stem borers. The siRNA nanoparticle biopharmaceutical utilizes polyethyleneimine and gold nanoparticles to effectively adsorb small interfering RNA (siRNA). The siRNA-adsorbed gold nanoparticles are then embedded in a copper-based organometallic framework (OMF), extending the release time of the siRNA. The gold nanoparticles then react with chitosan to form a chitosan membrane, which is then emulsified with polyether-modified heptamethyltrisiloxane to enhance its permeability and adsorption properties, ultimately producing the siRNA nanoparticle biopharmaceutical.

[0042] The small interfering RNA nanobiological agent of the present invention can be effectively transmitted within the trunk of a pine tree and can efficiently penetrate into the body of the pine alternating beetle, thereby effectively preventing and controlling the pine alternating beetle. Moreover, the small interfering RNA in the small interfering RNA nanobiological agent of the present invention has dual pH and enzyme responses and a good sustained-release effect, and has broad application prospects in the prevention and control of trunk-boring pests.

[0043] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0044] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. Experimental methods without detailed conditions were carried out in accordance with conventional test methods or the operating instructions recommended by the supplier.

[0045] The CAS number of polyethylene glycol is 25322-68-3; the CAS number of polyethyleneimine is 9002-98-6; the CAS number of chitosan is 9012-76-4.

[0046] The sequence of the small interfering RNA that interferes with the chitin deacetylase gene (MaCDA2b, GenBank: KY914480.1) of Monochamus alternatus is:

[0047] Sense:GCUCCUACGACGAUUGGUU(dT)(dT)

[0048] Antisense:AACCAUCGUCGUAGGAGC(dT)(dT)

[0049] Example 1: Preparation of small interfering RNA nanoparticles for controlling stem borers

[0050] (1) Add 100 mL of 0.01% (mass fraction) chloroauric acid solution to a three-necked round-bottom flask, place it in an oil bath at 155°C and stir magnetically for 20 min until the liquid boils. Add 3 mL of 1% (mass fraction) trisodium citrate solution and react for 20 min. When the solution turns bright red, remove it from the flask and place it in ice water to cool to room temperature to obtain gold nanoparticles.

[0051] (2) 7 mL of 0.5 mg / mL polyethylene glycol solution was added dropwise to the gold nanoparticles, magnetically stirred for 2.5 h, centrifuged (4°C, 14,000 rpm, 30 min), and washed three times with ultrapure water to remove excess polyethylene glycol to obtain polyethylene glycol-stabilized gold nanoparticles.

[0052] (3) Add 100 mL of a 1 mg / mL polyethyleneimine solution to the polyethylene glycol-stabilized gold nanoparticles. Ultrasonicate in an ice bath for 30 min. Centrifuge and wash three times with DEPC water to remove excess polyethyleneimine.

[0053] (4) Add 200 μg of small interfering RNA that interferes with the chitin deacetylase gene of Monochamus alternatus (MaCDA2b, GenBank: KY914480.1) to the system of step (3), sonicate in an ice bath in the dark for 30 min, centrifuge, and wash twice with DEPC water to remove free small interfering RNA.

[0054] (5) Add 100 mL of 1 mg / mL polyethyleneimine solution to the system of step (4) above, sonicate in an ice bath in the dark for 30 min, centrifuge, and wash three times with deionized water to obtain gold nanoparticle-loaded small interfering RNA nanopesticide.

[0055] (6) Dissolve 0.045g of copper nitrate and 0.01g of polyvinylpyrrolidone in 5mL of methanol and stir magnetically to obtain a first solution; dissolve 0.0215g of 1,3,5-benzenetricarboxylic acid in 5mL of methanol to obtain a second solution. Then, add the small interfering RNA nanopesticide loaded with gold nanoparticles prepared in step (5) to the first solution, mix well, and then add the second solution. Let it stand for 12 hours and collect the precipitate by centrifugation. The precipitate is washed with methanol three times for purification, transferred to the aqueous phase, and vacuum dried to prepare the small interfering RNA co-loaded with gold nanoparticles / organic metal framework, which is stored at 4°C for future use.

[0056] The prepared gold nanoparticles / organic metal framework co-loaded small interfering RNA was observed by transmission electron microscopy and scanning electron microscopy, and the results are as follows: Figure 1 shown.

[0057] The element distribution of the prepared gold nanoparticles / organic metal framework co-loaded small interfering RNA was detected, and the results were as follows Figure 2 shown.

[0058] (7) 10 mg of the gold nanoparticles / organic metal framework co-loaded small interfering RNA prepared in step (6) was dispersed into 25 mL of deionized water, and 10 mL of 1 g / L chitosan solution was added under magnetic stirring. The reaction was completed, and the precipitate was collected by centrifugation, purified with deionized water several times, and redispersed in 25 mL of deionized water. 1 mL of polyether-modified heptamethyltrisiloxane was added, and ultrasonic emulsification was performed for 30 minutes to prepare a small interfering RNA nanobiological agent for controlling stem borers.

[0059] Test Example 1:

[0060] The small interfering RNA nanopharmaceutical for controlling stem borers prepared in Example 1 was dispersed in PBS buffer solutions with pH values ​​of 6.0, 7.4, and 8.2 and incubated in an oscillating incubator at 15°C, 25°C, and 35°C. After a predetermined time, 200 μL of the sample suspension was centrifuged to obtain a supernatant. The siRNA concentration in the supernatant was measured using an ultramicrospectrophotometer, and the cumulative siRNA release efficiency was calculated.

[0061] The results are as follows Figure 3 As shown, the siRNA in the small interfering RNA nanopharmaceutical prepared by the present invention can achieve a sustained and controlled release effect. The entire sustained release process does not experience a sudden release. The sustained release rate is initially fast and then gradually slows down. The entire sustained release process reaches equilibrium in about 168 hours (7 days), and the release amount can reach 60% after 96 hours. The release of siRNA in the small interfering RNA nanopharmaceutical can last for 673 hours (28 days), indicating that the small interfering RNA nanopharmaceutical can achieve the effect of increasing the lasting effect.

[0062] The release of siRNA from the small interfering RNA nanopharmaceutical did not change significantly with temperature, indicating stable release in the environment. The release profile of siRNA under different pH conditions was pH = 7.4 > 8.2 > 6.0, demonstrating that the siRNA in the small interfering RNA nanopharmaceutical prepared by the present invention can be released in response to pH conditions. Furthermore, the polyethyleneimine in the small interfering RNA nanopharmaceutical is enzymatically degraded, resulting in responsive release in insects. This indicates that the small interfering RNA nanopharmaceutical prepared by the present invention exhibits both pH- and enzyme-responsive controlled siRNA release.

[0063] Test Example 2:

[0064] In order to investigate the effect of the small interfering RNA nanoparticle biopharmaceutical prepared by the present invention on Monochamus alternatus larvae, the small interfering RNA in Example 1 was fluorescently labeled (NC-FAM-siRNA) and the small interfering RNA nanoparticle biopharmaceutical was prepared according to the method of Example 1.

[0065] The prepared small interfering RNA nanoparticles were sprayed on the body surface of the fourth-instar pine beetle larvae. The coverage, permeability and adhesion of the small interfering RNA nanoparticles on the pine beetle larvae were observed using a stereo fluorescence microscope. The siRNA absorption and the distribution performance of the small interfering RNA nanoparticles were determined by observing their fluorescence distribution.

[0066] Fluorescence images of small interfering RNA nanopharmaceuticals Figure 4 As shown in Figure A, the small interfering RNA nanoparticles exhibited distinct green fluorescence, demonstrating successful siRNA encapsulation. Stereofluorescence microscopy revealed distinct and uniform green fluorescence on the epidermis of the treated Monochamus alternatus larvae, distributed throughout the entire larval body wall. The fluorescence was more pronounced in the intersegmental membranes of the body wall than in other parts ( Figure 4 B). At the same time, there are multiple independent bright fluorescent particles distributed around the stomata on the side of the larvae. The magnified image can more clearly observe the strong green fluorescence distributed around the stomata ( Figure 4 C) Observe the fluorescence distribution in the intestinal tissue to study the distribution of small interfering RNA nanopharmaceuticals in the intestine, such as Figure 4 As shown in D, the entire outline of the intestinal tissue in the cross-section of the midgut of the Monochamus alternatus larvae is evenly distributed with fluorescence, indicating that the nano-siRNA complex can smoothly reach the intestinal tissue.

[0067] The above results show that the small interfering RNA nanobiological agent prepared by the present invention can encapsulate the interfering chitin deacetylase gene of pine sawyer beetle (MaCDA2b-siRNA), and adsorb and highly permeate into the insect body, thereby achieving high permeability delivery of "trunk pest pine sawyer beetle body wall-target cell".

[0068] Test Example 3:

[0069] This test example simulates the actual transmission performance of forest pesticide application by analyzing the diffusion of the small interfering RNA nanopharmaceutical prepared by the present invention in the tree body.

[0070] Pine trees aged 20-30 years and with a uniform diameter of approximately 15 cm were selected for the experiment. Small interfering RNA nanoparticles containing fluorescently labeled NC-Cy3-siRNA were injected at different concentrations (20, 40, 60, and 80 μg / 10 mL) per tree using traditional punch injection. The transmission of small interfering RNA nanoparticles encapsulating fluorescently labeled NC-Cy3-siRNA at different concentrations (20, 40, 60, and 80 μg / 10 mL) at different observation angles (cross-section, injection surface, and longitudinal section) of the tree was observed using an in vivo imaging system to screen out the concentration of small interfering RNA nanoparticles used in the transmission experiment. Then, the small interfering RNA nanobiological agent encapsulated with fluorescently labeled NC-Cy3-siRNA was injected into the tree body through the traditional punch injection method. The pine trees were observed after different treatment times (1, 3, 5, and 7 hours) and the pine wood was cut into blocks along the pine pith. The lateral and longitudinal transmission of NC-Cy3-siRNA in the nano-siRNA complex within the pine tree body was observed using a live imaging system.

[0071] The transport of small interfering RNA nanoparticles with different concentrations (20, 40, 60, 80 μg / 10 mL) in pine trees is shown in Figure 2. Figure 5 As shown, strong fluorescence distribution was present at all concentrations, and the fluorescence gradually increased with increasing concentration (20, 40, 60, and 80 μg / 10 mL), with the strongest fluorescence in the xylem. This indicates that the small interfering RNA nanoparticle has good dispersion and transmission properties in pine trees. In subsequent studies on the horizontal and vertical transmission of small interfering RNA nanoparticles in trees, the concentration used was 40 μg / 10 mL.

[0072] 40 μg / 10 mL of a small interfering RNA nanoparticle biopharmaceutical encapsulated with fluorescently labeled NC-Cy3-siRNA was injected into the tree body by traditional punch injection. After different treatment times (1, 3, 5, and 7 hours), the pine trees were observed and the pine wood was cut into pieces along the pith. The lateral and longitudinal transport of NC-Cy3-siRNA in the small interfering RNA nanoparticle biopharmaceutical within the pine tree body was observed using an in vivo imaging system. Figure 6As shown, strong fluorescence distribution was present at different times, and the fluorescence increased over time (1, 3, 5, and 7 hours). The horizontal and vertical fluorescence of the tree gradually increased, and strong fluorescence was dispersed throughout the xylem of the tree. This indicates that the small interfering RNA nanopharmaceutical prepared in this experiment has good dispersion and transmission properties within the tree. The small interfering RNA nanopharmaceutical can achieve high permeability into the tree by encapsulating the small interfering RNA that interferes with the chitin deacetylase gene of the pine alternating beetle, achieving a "tree-trunk pest pine alternating beetle body wall" effect. This also proves that this small interfering RNA nanopharmaceutical has certain development prospects in the practical application of controlling pine alternating beetles.

[0073] Test Example 4:

[0074] The small interfering RNA nanopharmaceutical prepared in Example 1 was prepared into three different concentrations: 4 μg / mL, 8 μg / mL, and 16 μg / mL; and divided into contact treatment (CT) and stomach poisoning treatment (ST) according to the application mode; wherein:

[0075] The contact treatment was to spray equal volumes of 4 μg / mL, 8 μg / mL, and 16 μg / mL small interfering RNA nanopharmaceuticals at different concentrations on the surface of the fourth-instar pine beetle larvae, and spraying equal volumes of clean water was used as the control (CK).

[0076] The stomach poisoning treatment was to inject equal volumes of 4μg / mL, 8μg / mL, and 16μg / mL small interfering RNA nanopharmaceuticals at three different concentrations into the fourth-instar pine beetle larvae, and the injection of equal volumes of water was used as the control (CK).

[0077] After 72 h of treatment, the relative expression level of MaCDA2b gene (GenBank: KY914480.1) was determined by fluorescence quantitative analysis.

[0078] The results are as follows Figure 7 As shown, the relative expression of the MaCDA2b gene in Monochamus alternatus larvae was downregulated to varying degrees when treated with different concentrations of small interfering RNA nanopharmaceuticals (4 μg / mL, 8 μg / mL, and 16 μg / mL), with the higher the concentration, the more significant the inhibitory effect. At the same concentration of small interfering RNA nanopharmaceuticals, the relative expression of the MaCDA2b gene was lower in the stomach-poisoning group than in the contact-killing group.

[0079] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A small interfering RNA nanobiological agent, characterized in that: Prepared by the following method: (1) mixing a polyethyleneimine solution and polyethylene glycol-stabilized gold nanoparticles, adding small interfering RNA, and then adding the polyethyleneimine solution to obtain a gold nanoparticle-loaded small interfering RNA nanopesticide; (2) dissolving copper nitrate and polyvinyl pyrrolidone in methanol to obtain a first solution, and dissolving 1,3,5-benzenetricarboxylic acid in methanol to obtain a second solution; adding the small interfering RNA nanopesticide loaded with gold nanoparticles to the first solution, mixing well, and then adding the second solution, standing, centrifuging, collecting the precipitate, washing, and drying to prepare the small interfering RNA co-loaded with gold nanoparticles / organic metal framework; (3) The small interfering RNA co-loaded by the gold nanoparticles / organic metal framework is dispersed in deionized water, chitosan solution is added, and the precipitate is collected by centrifugation after the reaction. The precipitate is redispersed in deionized water, polyether-modified heptamethyltrisiloxane is added, and ultrasonic emulsification is performed to prepare the small interfering RNA nanobiological agent.

2. The small interfering RNA nanobiological agent according to claim 1, characterized in that In step (1), the concentration of the polyethyleneimine solution is 0.1-2 mg / mL.

3. The small interfering RNA nanobiological agent according to claim 1, characterized in that In step (1), the polyethylene glycol-stabilized gold nanoparticles are prepared by the following method: The polyethylene glycol solution was added dropwise to the gold nanoparticles, stirred for 2-3 hours, centrifuged, and washed to prepare polyethylene glycol-stabilized gold nanoparticles.

4. The small interfering RNA nanobiological agent according to claim 1, characterized in that In step (1), the small interfering RNA is a small interfering RNA targeting growth and development genes, reproductive genes and / or olfactory genes of stem-boring pests; Preferably, the small interfering RNA targets the Monochamus alternatus chitin deacetylase gene MaCDA2b.

5. The small interfering RNA nanobiological agent according to claim 1, characterized in that In step (2), the concentration of copper nitrate in the first solution is 0.005 g / mL-0.015 g / mL, and the concentration of polyvinyl pyrrolidone is 0.001 g / mL-0.003 g / mL; the concentration of 1,3,5-benzenetricarboxylic acid in the second solution is 0.001 g / mL-0.01 g / mL; and the volume ratio of the first solution to the second solution is 1:

1.

6. The small interfering RNA nanobiological agent according to claim 1, characterized in that: In step (3), the mass ratio of the small interfering RNA co-loaded by the gold nanoparticles / organic metal framework to chitosan is 1:10-10:1; the mass ratio of the small interfering RNA co-loaded by the gold nanoparticles / organic metal framework to polyether-modified heptamethyltrisiloxane is 1:100-1000:

1.

7. The small interfering RNA nanobiological agent according to claim 1, characterized in that In step (3), the ultrasonic reaction time is 10-120 min.

8. Use of the small interfering RNA nanobiological agent according to any one of claims 1 to 7 in the following (1) or (2): (1) Prevent and control stem-boring pests; (2) Preparation of products for controlling stem borers.

9. The use according to claim 8, characterized in that The trunk-boring pests include longhorn beetles, wood borers, jellyfish, bark beetles and weevils.

10. A method for controlling stem-boring pests, characterized in that: The following steps are involved: Before or when the trunk-boring pests become ill, the small interfering RNA nanobiological agent according to any one of claims 1 to 7 is sprayed on the surface of the tree trunk.

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