DsRNA nanoparticle complex of a trehalase gene for controlling citrus psyllid and preparation method thereof

Abstract

The application belongs to the technical fields of agricultural pest control and nucleic acid biological pesticide, and discloses a trehalase gene double-stranded RNA nanoparticle complex for preventing and treating citrus psylla and a preparation method thereof.The complex is composed of 100-120 parts of insecticide, 8-12 parts of modified graphene oxide nanocarrier and 4-6 parts of double-stranded RNA targeting trehalase gene according to weight parts.The application prepares high-specificity double-stranded RNA through in vitro transcription method or in vivo expression method of HT115 engineering bacteria, constructs a nanocarrier drug system by using the electrostatic self-assembly characteristics of modified graphene oxide, and realizes the efficient protection of double-stranded RNA and the synergistic compounding of insecticide by combining high-frequency acoustic field treatment and high-shear dispersion technology.The application has the advantages of high target specificity and ecological safety, excellent dsRNA environmental stability and anti-degradation ability, significant synergistic effect and resistance overcoming effect, excellent loading efficiency and transmembrane delivery capacity, flexible and efficient production process and industrialization potential.

Description

Technical Field

[0001] This invention discloses a trehalase gene dsRNA nanoparticle complex for controlling citrus psyllids and its preparation method, belonging to the field of agricultural pest control and nucleic acid biopesticide technology. Background Technology

[0002] The citrus psyllid (Diaphorina citri Kuwayama) is a core pest of citrus crops. It not only directly sucks sap from young shoots, causing leaf deformities and inducing sooty mold, but also serves as the sole natural vector for Huanglongbing (HLB), the world's most destructive citrus disease, causing yield reduction and even orchard death through persistent transmission of the virus. Currently, control of the citrus psyllid still heavily relies on the frequent application of chemical pesticides. However, long-term and excessive use of conventional chemical agents has not only led to significant pesticide resistance in the psyllid population, drastically reducing control effectiveness, but also negatively impacted beneficial insects, pollinators, and the surrounding ecological balance due to the broad-spectrum toxicity of these pesticides. Therefore, developing novel biological control technologies with high specificity, environmental friendliness, and effectiveness against multiple resistant psyllids has become an urgent need for the citrus industry.

[0003] RNA interference (RNAi), a biotechnology that uses double-stranded RNA (dsRNA) to specifically induce the degradation of target mRNA and thereby silence key life genes, has shown great potential in the field of precision pest control. Trehalase (HK) is an important hydrolytic enzyme in insects, responsible for converting trehalose into glucose, and directly involved in the energy metabolism, molting process, chitin synthesis, and growth and development regulation of pests. Previous studies have shown that interfering with the expression of the HK gene in psyllids can lead to blood glucose imbalance, resulting in molting disorders or death. However, the application of dsRNA in the field faces significant technical challenges: on the one hand, ultraviolet radiation, temperature, and rainfall in the external environment can easily inactivate it; on the other hand, ribonucleases (RNases) commonly found in the midgut and leaf surface of psyllids can rapidly degrade naked dsRNA, resulting in low bioavailability and difficulty in achieving ideal control stability.

[0004] To overcome the drawbacks of insufficient stability and difficult transmembrane absorption of dsRNA, physical or chemical protection using nanodelivery systems has become a current research hotspot. Graphene oxide, as a two-dimensional carbon nanomaterial with ultra-large specific surface area and excellent chemical regulation, shows great promise in the field of pesticide loading. However, unmodified raw graphene oxide is prone to aggregation in electrolyte environments, and electrostatic repulsion exists between it and negatively charged dsRNA molecules, limiting its loading efficiency. By carboxylating graphene oxide, modifying it with polyethylene glycol (PEG), and grafting it with polyethyleneimine (PEI), a ternary topological nanostructure with a steric hindrance protection layer and a positively charged adsorption center was constructed, which significantly improved the encapsulation ability of dsRNA and the delivery depth within the psyllid. Based on this, a preparation process for synergistically combining HK gene dsRNA with modified nanocarriers and highly efficient insecticides was developed, which has important scientific value and practical significance for constructing an efficient and long-lasting precision control system for citrus psyllids. Summary of the Invention

[0005] The purpose of this invention is to overcome the technical shortcomings of existing chemical pesticides for controlling citrus psyllids, such as strong pesticide resistance, significant environmental pollution, and poor stability and low bioavailability of nucleic acid-based biological pesticides. This invention provides a trehalase (HK) gene dsRNA nanoparticle complex for controlling citrus psyllids and its preparation method. This complex utilizes a modified graphene oxide nanocarrier to efficiently load and environmentally shield a specific sequence of dsRNA, combining this with the synergistic effect of insecticides to achieve precise control of citrus psyllids and effective blocking of the spread of citrus Huanglongbing (HLB).

[0006] To achieve the purpose of this invention, the technical solution adopted is as follows: A nanoparticle complex of double-stranded RNA targeting the trehalase gene of citrus psyllid, comprising, by weight, the following components: 100-120 parts insecticide, 8-12 parts modified graphene oxide nanocarrier, and 4-6 parts double-stranded RNA targeting the trehalase gene of citrus psyllid; wherein the double-stranded RNA targeting the trehalase gene of citrus psyllid is prepared by either pathway A or pathway B: Path A: S1 Collect adult citrus psyllids and put them into an electric grinder. Add liquid nitrogen and grind at 1500-1600 rpm until a grayish-white powder with no visible tissue is formed. Add acidic lysis buffer to the powder and let it stand at 15-25℃ for 5-10 min to destroy the cell membrane structure and inactivate endogenous ribonuclease using the liquid salt. Add 0.2-0.3 times the volume of chloroform to the lysis system, shake at 4000-5000 rpm for 10-15 s, and then centrifuge at 12000-13000 rpm for 15-20 min at 4-6℃. The colorless and transparent upper aqueous phase was extracted and transferred to a nuclease-free centrifuge tube. An equal volume of isopropanol was added, and the mixture was incubated at -20 to -18°C for 30 to 40 minutes to drive the dehydration and precipitation of RNA molecules. After centrifugation at 12,000 to 13,000 rpm for 10 to 20 minutes, the supernatant was discarded. The precipitate was washed twice with ethanol solution to remove residual salts and organic solvents. The RNA precipitate was dissolved in 10 to 20 μL of ultrapure nuclease-free water to obtain a high-abundance total RNA matrix. S2. Take total RNA, add deoxyribonuclease I, and treat at 37-39℃ for 10-20 min to completely eliminate interference from genomic DNA; add oligodeoxythymidine primers, heat at 65-75℃ for 5-10 min to expand the secondary structure of RNA, and then quickly place on ice to cool; add reverse transcriptase, deoxyribonucleoside triphosphate mixture and reaction buffer, extend at 42-44℃ for 60-120 min, and then heat at 70-80℃ for 15-25 min to inactivate reverse transcriptase, finally obtaining full-length single-stranded cDNA containing the trehalase gene of citrus psyllid; S3 uses the primer pair shown in SEQ ID NO: 2 and SEQ ID NO: 3. Using the obtained cDNA as a template, DNA polymerase is added. The mixture is pre-denatured at 95-96℃ for 3-4 min; denatured at 98-100℃ for 10-20 s; annealed at 58-60℃ for 20-30 s; extended at 72-74℃ for 30-60 s, for a total of 35-40 cycles; and finally extended at 70-72℃ for 5-10 min. The amplified product is subjected to agarose gel electrophoresis, and the target fragment is obtained using gel extraction technology, which serves as the template DNA for in vitro transcription and synthesis of double-stranded RNA targeting the trehalase gene. In step S4, the obtained template DNA, ATP, CTP, GTP, UTP, T7 RNA polymerase, and ribonuclease inhibitor were added to a centrifuge tube. The reaction system was incubated in a constant temperature dry bath at 37–40°C for 8–12 hours. After the reaction, the system was heated to 75–80°C and held for 5–10 minutes to eliminate non-specific pairing. The system was then cooled to 20–25°C at a forced cooling rate of 1.5–2°C per minute. During this process, single-stranded RNA molecules achieve precise base pairing through intermolecular hydrogen bonds, resulting in double-stranded RNA molecules with a specific spatial conformation targeting the trehalase gene. Path B: S5 used primers with restriction enzyme sites shown in SEQ ID NO: 6 and SEQ ID NO: 7 to amplify the trehalase fragment targeting the citrus psyllid. The fragment was then inserted into the multiple cloning site of the L4440 plasmid using T4 DNA ligase to obtain a recombinant plasmid. The recombinant plasmid was transformed into competent HT115(DE3) *E. coli* cells. The transformed strain was cultured in a medium at 37–40°C and 220–240 rpm. When the absorbance of the bacterial culture reached 0.4–0.6, isopropyl-β-D-thiogalactoside was added to a final concentration of 0.8–1.0 mM. The fermentation temperature was lowered to 28–30°C, and fermentation was induced for 8–12 hours. The bacterial pellet was collected and subjected to repeated freeze-thaw cycles combined with heat pulses to disrupt the bacterial cell wall and membrane structure, releasing the intracellularly synthesized double-stranded RNA targeting the citrus psyllid trehalase gene. The pellet was then subjected to 12,000–13,000 [units of temperature missing]. After centrifugation at rpm to remove impurities, the double-stranded RNA targeting the trehalase gene of the citrus psyllid was finally obtained.

[0007] According to a double-stranded RNA nanoparticle complex of trehalase gene for controlling citrus psyllids, the insecticide is one or more of imidacloprid, thiamethoxam, acetamiprid, and etoxazole.

[0008] According to a double-stranded RNA nanoparticle complex of trehalase gene for the control of citrus psyllids, the acidic lysis buffer described in S1 is one or more of guanidine isothiocyanate, sodium dodecyl sarcosinate, and phenol.

[0009] According to a double-stranded RNA nanoparticle complex of trehalase gene for the control of citrus psyllids, the DNA polymerase is one or more of Pfu DNA polymerase, KOD DNA polymerase, and Taq DNA polymerase.

[0010] According to a double-stranded RNA nanoparticle complex of trehalase gene for the control of citrus psyllids, the ribonuclease inhibitor is one or more of the following: recombinant placental ribonuclease inhibitor, diethyl pyrocarbonate, vanadium ribonucleoside complex, guanidine isothiocyanate, and sodium dodecyl sarcosinate.

[0011] According to a double-stranded RNA nanoparticle complex of trehalase gene for the control of citrus psyllids, the modified graphene oxide nanocarrier is a ternary topological nanosheet constructed by covalently grafting carboxylated graphene oxide with polyethylene glycol and polyethyleneimine, which has a positively charged surface and a steric hindrance protection layer.

[0012] According to a double-stranded RNA nanoparticle complex of trehalase gene for controlling citrus psyllids, the preparation process of the double-stranded RNA nanoparticle complex of trehalase gene for controlling citrus psyllids includes the following steps: S6 mixes double-stranded RNA solution with modified graphene oxide nanocarrier solution at a volume ratio of 1:2~3, and stirs at 100~200 rpm for 30~50 min at 20~25℃ to construct primary complex through electrostatic self-assembly. S7 The obtained primary complex was transferred to a high-frequency energy processing chamber and treated for 15-20 min under an acoustic field with a frequency of 40-60 kHz and a power density of 1.8-2.5 W / cm². The non-covalent aggregation between double-stranded RNA, polymer auxiliary components and nanosheets was eliminated through acoustic cavitation effect. S8 The treated complex is pumped into the insecticide solution at a volume ratio of 1:10~15, and circulated and cut at a speed of 3000~5000 rpm for 10~20 min to make the insecticide molecules form a molecular-level dispersed distribution on the surface of the complex. The mixture is then cooled to room temperature to obtain the final product. Beneficial effects

[0013] (1) Extremely high targeting specificity and ecological safety: The dsHK fragment screened in this invention has high specificity against the trehalase gene of the citrus psyllid, selectively interfering only with the energy metabolism and development process of the target pest, and is completely harmless to beneficial agricultural insects such as bees, predatory mites, and ladybugs. Compared with traditional broad-spectrum insecticides, this invention effectively protects the biodiversity of orchard ecosystems through gene-level specific targeting.

[0014] (2) Excellent environmental stability and anti-degradation ability of dsRNA: By covalently grafting polyethylene glycol (PEG) and polyethyleneimine (PEI) onto the surface of graphene oxide, a nanotransport system with dual protection effects of "electrostatic shielding + steric hindrance" was constructed. This carrier can tightly encapsulate dsRNA through charge chelation, significantly resisting external ultraviolet radiation, high temperature fluctuations, and degradation by RNase enzymes on the midgut of insects and leaf surfaces, thus solving the key technical bottleneck of short field persistence of dsRNA.

[0015] (3) Significant synergistic effect and resistance overcoming: This invention utilizes a dual mechanism of "gene silencing + chemical poisoning" to significantly increase the sensitivity of citrus psyllids to insecticides such as imidacloprid and thiamethoxam under physiological conditions of impaired energy metabolism and disordered sugar metabolism. This synergistic effect not only greatly reduces the amount of conventional chemical pesticides used (reducing environmental pressure), but also effectively solves the serious pesticide resistance problem caused by long-term pesticide use in citrus psyllids.

[0016] (4) Excellent loading efficiency and transmembrane delivery capability: The ternary topological structure nanocarrier has an ultra-large specific surface area and obvious positive charge adsorption sites, which can achieve high loading rate of dsRNA. At the same time, the PEI component has excellent membrane affinity, which can help dsRNA penetrate the epidermal and intestinal mucosal barriers of pests, significantly improving the bioavailability of RNAi molecules.

[0017] (5) Flexible and efficient production process and industrialization potential: The pathway A (high-purity in vitro transcription) and pathway B (low-cost cell fermentation) provided in the invention complement each other, and the production scale can be flexibly adjusted according to market demand. In addition, the acoustic field dissipation (S7) and high shear dispersion (S8) technologies introduced in the process ensure the uniform distribution of the complex at the molecular level, effectively prevent the non-valent aggregation of nanoparticles, and ensure the high stability of product quality. Attached Figure Description

[0018] Figure 1 To validate the dsHK-L4440 recombinant plasmid; Figure 2 Results of dsHK expression in Escherichia coli; Figure 3 To investigate the expression of dsHK synthesized in vitro and expressed in Escherichia coli in citrus psyllids; Figure 4 The agarose gel prevented the experimental results; Figure 5 This is a schematic diagram of the indoor bioassay measurement method; Figure 6 Changes in the survival rate of citrus psyllids treated with imidacloprid / GONs-dsHK. Detailed Implementation

[0019] Example 1

[0020] To synthesize double-stranded RNA targeting key physiological genes of the citrus psyllid, this invention first extracted total RNA from adult citrus psyllids using the Trizol method and reverse transcribed it to synthesize a single-stranded cDNA matrix. Based on the existing coding sequence of the trehalase (HK) gene, specific primers (SEQ ID NO: 2 and SEQ ID NO: 3) with the T7 promoter sequence were designed and synthesized, and the target fragment was amplified using PCR technology. Figure 1 Using the amplified product as a template, dsHK molecules were synthesized on a large scale using an in vitro transcription kit. To further reduce production costs, the L4440 / HT115(DE3) in vivo expression system was also used. The HK fragment was constructed into a recombinant plasmid using primers with restriction sites (SEQ ID NO: 6 and SEQ ID NO: 7) and transformed into RNase III-deficient E. coli, followed by expression induction with 1 mM IPTG. Electrophoresis analysis results ( Figure 2 The results showed that undigested E. coli total RNA had no obvious band in lane 1, while lane 2, after digestion, showed a clear target band at approximately 500 bp. This demonstrates that the E. coli expression system can successfully synthesize biologically active dsHK molecules.

[0021] Before constructing the nanocomposite, this invention utilized electron microscopy to characterize the microstructure of the modified graphene oxide nanocarriers (GONs). For example... Figure 3 As shown, the modified GONs exhibit a typical two-dimensional sheet-like topology, with their surface displaying fine wrinkles due to the covalent grafting of polyethylene glycol (PEG) and polyethyleneimine (PEI). This unique sheet-like morphology not only endows the carrier with a large specific surface area, but also provides an ideal physical support site for the subsequent efficient adsorption of dsRNA and the molecular-level dispersion of pesticide molecules.

[0022] In subsequent assembly experiments, GONs were diluted 50-fold and then thoroughly mixed with 2000 ng / μL dsHK solution at a 2:1 volume ratio and incubated. Utilizing charge interactions and steric hindrance, dsHK was firmly locked within the nanosheet structure. Gel electrophoresis retardation experiments were then performed. Figure 4 The results showed that after dsHK and GONs formed a complex, no downstream dsRNA migration bands were observed in the gel image for two consecutive days, even when mixed with imidacloprid insecticide; all nucleic acid molecules remained at the sample wells. This fully demonstrates that GONs have a very high affinity and protective effect on dsHK, preventing it from dissociating or being prematurely degraded in complex insecticide systems.

[0023] To evaluate the bioactivity and gene silencing efficiency of the complex of this invention, ten adult citrus psyllids, pre-starved for 24 hours, were fed on citrus leaves containing 9 μg of dsHK. The expression level of the HK gene in the pests was detected using real-time quantitative PCR (qRT-PCR) and specific primers (SEQ ID NO: 4 and SEQ ID NO: 5). Analysis results ( Figure 5The results showed that feeding nanoparticles loaded with dsHK significantly suppressed the expression of the HK gene in citrus psyllids. The dsHK obtained from in vivo expression in *E. coli* exhibited a highly efficient silencing effect consistent with the in vitro transcription product, demonstrating that the nucleic acid molecules prepared via this pathway can fully meet the needs for gene interference in practical production.

[0024] Based on this, the present invention constructs a synergistic insecticidal system by dissolving 0.2 g of imidacloprid powder and mixing it with 10 mL of GONs-dsHK stock solution. To verify whether this system can significantly improve the sensitivity of citrus psyllids to the pesticide, researchers designed multiple experimental groups, including a dsGFP control, imidacloprid alone, imidacloprid / GONs-dsGFP, and the imidacloprid / GONs-dsHK described in this invention. The lethality of 50 adults in each group was recorded over 48 hours. Toxicity test results ( Figure 6 The study clearly indicated that the survival rate of citrus psyllids remained at a high level in all dsGFP control groups and single-agent groups. However, the survival rate of adults in the imidacloprid / GONs-dsHK group plummeted 12 hours after application, significantly lower than that of other control treatments. This result not only confirms that downregulation of the trehalase gene accelerates the mortality process of the pests, but also demonstrates that nanocarrier-mediated dsRNA can significantly enhance the efficacy of insecticides at extremely low dose levels, providing strong support for the precise control of citrus psyllids.

[0025] Example 2

[0026] Adult citrus psyllids were collected and placed in an electric grinder. Liquid nitrogen was added, and the mixture was ground at 1500 rpm until it became a grayish-white powder with no visible tissue. Acidic lysis buffer was added to the powder, and the mixture was allowed to stand at 15°C for 5 minutes. The liquid salt was used to disrupt the cell membrane structure and inactivate endogenous ribonuclease. 0.2 times the volume of chloroform was added to the lysis system, and the mixture was shaken at 4000 rpm for 10 seconds. Then, it was centrifuged at 12000 rpm for 15 minutes at 4°C. The colorless, transparent upper aqueous phase was extracted and transferred to a nuclease-free centrifuge tube. An equal volume of isopropanol was added, and the mixture was incubated at -20°C for 30 min to drive RNA dehydration and precipitation. After centrifugation at 12,000 rpm for 10 min, the supernatant was discarded. The precipitate was washed twice with ethanol solution to remove residual salts and organic solvents. The RNA precipitate was dissolved in 10 μL of ultrapure nuclease-free water to obtain a high-abundance total RNA matrix. The total RNA was then treated with deoxyribonuclease I at 37°C for 10 min to completely eliminate interference from genomic DNA. Oligodeoxythymidine primers were added, and the mixture was heated at 65°C for 5 min to expand the RNA secondary structure, followed by rapid cooling on ice. Reverse transcriptase, a mixture of deoxyribonucleoside triphosphates, and reaction buffer were added, and the mixture was extended at 42°C for 60 min, followed by heating at 70°C for 15 min to inactivate the reverse transcriptase, finally obtaining a full-length single-stranded cDNA containing the trehalase gene from the citrus psyllid. SEQ ID NO: Primers 2 and SEQ ID NO: 3 were used as templates. DNA polymerase was added, and the mixture was pre-denatured at 95°C for 3 min, followed by denaturation at 98°C for 10 s, annealing at 58°C for 20 s, and extension at 72°C for 30 s, for a total of 35 cycles. Finally, the mixture was fully extended at 70°C for 5 min. The amplified products were subjected to agarose gel electrophoresis, and the target fragment was obtained using gel extraction technology. This fragment was used as template DNA for in vitro transcription and synthesis of double-stranded RNA targeting the trehalase gene. The obtained template DNA, ATP, CTP, GTP, UTP, T7 RNA polymerase, and ribonuclease inhibitor were added to centrifuge tubes, and the reaction system was incubated in a 37°C dry bath for 8 hours. After the reaction, the system was heated to 75°C and held for 5 min to eliminate non-specific pairing. The system was then cooled to 20°C at a forced cooling rate of 1.5°C per minute. In this process, single-stranded RNA molecules achieve precise base pairing through intermolecular hydrogen bonds, resulting in double-stranded RNA molecules with a specific spatial conformation that target the trehalase gene.

[0027] Example 3

[0028] Adult citrus psyllids were collected and placed in an electric grinder. Liquid nitrogen was added, and the mixture was ground at 1550 rpm until it became a grayish-white powder with no visible tissue. Acidic lysis buffer was added to the powder, and the mixture was allowed to stand at 20°C for 7 minutes. The liquid salt was used to disrupt the cell membrane structure and inactivate endogenous ribonuclease. 0.2 times the volume of chloroform was added to the lysis system, and the mixture was shaken at 4500 rpm for 12 seconds. Then, it was centrifuged at 12500 rpm for 17 minutes at 5°C. The colorless, transparent upper aqueous phase was extracted and transferred to a nuclease-free centrifuge tube. An equal volume of isopropanol was added, and the mixture was incubated at -19°C for 35 min to drive RNA dehydration and precipitation. After centrifugation at 12500 rpm for 15 min, the supernatant was discarded. The precipitate was washed twice with ethanol solution to remove residual salts and organic solvents. The RNA precipitate was dissolved in 15 μL of ultrapure nuclease-free water to obtain a high-abundance total RNA matrix. The total RNA was then treated with deoxyribonuclease I at 38°C for 15 min to completely eliminate interference from genomic DNA. Oligodeoxythymidine primers were added, and the mixture was heated at 70°C for 7 min to expand the RNA secondary structure, followed by rapid cooling on ice. Reverse transcriptase, a mixture of deoxyribonucleoside triphosphates, and reaction buffer were added, and the mixture was extended at 43°C for 90 min, followed by heating at 75°C for 20 min to inactivate the reverse transcriptase, finally obtaining a full-length single-stranded cDNA containing the trehalase gene from the citrus psyllid. SEQ ID NO: Primers 2 and SEQ ID NO: 3 were used as templates. DNA polymerase was added, and the mixture was pre-denatured at 95°C for 3 min, followed by denaturation at 99°C for 15 s, annealing at 59°C for 25 s, and extension at 73°C for 45 s, for a total of 37 cycles. Finally, the mixture was fully extended at 71°C for 7 min. The amplified products were subjected to agarose gel electrophoresis, and the target fragment was obtained using gel extraction technology. This fragment was used as template DNA for in vitro transcription to synthesize double-stranded RNA targeting the trehalase gene. The obtained template DNA, ATP, CTP, GTP, UTP, T7 RNA polymerase, and ribonuclease inhibitor were added to centrifuge tubes, and the reaction system was incubated in a 38°C dry bath for 10 hours. After the reaction, the system was heated to 77°C and held for 7 min to eliminate non-specific pairing. The system was then cooled to 22°C at a forced cooling rate of 1.5°C per minute. In this process, single-stranded RNA molecules achieve precise base pairing through intermolecular hydrogen bonds, resulting in double-stranded RNA molecules with a specific spatial conformation that target the trehalase gene.

[0029] Example 4

[0030] Adult citrus psyllids were collected and placed in an electric grinder. Liquid nitrogen was added, and the mixture was ground at 1600 rpm until it became a grayish-white powder with no visible tissue. Acidic lysis buffer was added to the powder, and the mixture was allowed to stand at 25°C for 10 min to disrupt the cell membrane structure and inactivate endogenous ribonucleases. 0.3 times the volume of chloroform was added to the lysis system, and the mixture was shaken at 5000 rpm for 15 s. Then, it was centrifuged at 6°C and 13000 rpm for 20 min. The colorless, transparent upper aqueous phase was extracted and transferred to a nuclease-free centrifuge tube. An equal volume of isopropanol was added, and the mixture was incubated at -18°C for 40 min to drive RNA dehydration and precipitation. After centrifugation at 13000 rpm for 20 min, the supernatant was discarded. The precipitate was washed twice with ethanol solution to remove residual salts and organic solvents. The RNA precipitate was dissolved in 20 μL of ultrapure nuclease-free water to obtain a high-abundance total RNA matrix. The total RNA was then treated with deoxyribonuclease I at 39°C for 20 min to completely eliminate interference from genomic DNA. Oligodeoxythymidine primers were added, and the mixture was heated at 75°C for 10 min to expand the RNA secondary structure, followed by rapid cooling on ice. Reverse transcriptase, a mixture of deoxyribonucleoside triphosphates, and reaction buffer were added, and the mixture was extended at 44°C for 120 min, followed by heating at 80°C for 25 min to inactivate the reverse transcriptase, finally obtaining a full-length single-stranded cDNA containing the trehalase gene of the citrus psyllid. SEQ ID NO: Primers 2 and SEQ ID NO: 3 were used as templates. DNA polymerase was added, and the mixture was pre-denatured at 96℃ for 4 min, followed by denaturation at 100℃ for 20 s, annealing at 60℃ for 20-30 s, and extension at 74℃ for 60 s, for a total of 40 cycles. Finally, the mixture was fully extended at 72℃ for 10 min. The amplified products were subjected to agarose gel electrophoresis, and the target fragment was obtained using gel extraction technology. This fragment was used as template DNA for in vitro transcription to synthesize double-stranded RNA targeting the trehalase gene. The obtained template DNA, ATP, CTP, GTP, UTP, T7 RNA polymerase, and ribonuclease inhibitor were added to centrifuge tubes, and the reaction system was incubated in a 40℃ dry bath for 12 hours. After the reaction, the system was heated to 80℃ and held for 10 min to eliminate non-specific pairing. The system was then cooled to 25℃ at a forced cooling rate of 2℃ per minute. In this process, single-stranded RNA molecules achieve precise base pairing through intermolecular hydrogen bonds, resulting in double-stranded RNA molecules with a specific spatial conformation that target the trehalase gene.

[0031] Example 5

[0032] Primers with restriction enzyme sites as shown in SEQ ID NO: 6 and SEQ ID NO: 7 were used to amplify the trehalase fragment targeting the citrus psyllid. The fragment was then inserted into the multiple cloning site of the L4440 plasmid using T4 DNA ligase to obtain a recombinant plasmid. The recombinant plasmid was transformed into competent HT115(DE3) Escherichia coli cells. The transformed strain was cultured in a medium at 37°C and 220 rpm. When the absorbance of the bacterial culture reached 0.4, isopropyl-β-D-thiogalactoside was added to a final concentration of 0.8 mM. The fermentation temperature was lowered to 28°C, and fermentation was induced for 8 hours. The bacterial pellet was collected, and repeated freeze-thaw cycles combined with heat pulse treatment were used to disrupt the bacterial cell wall and membrane structure, releasing the intracellularly synthesized double-stranded RNA targeting the citrus psyllid trehalase gene. After centrifugation at 12000 rpm to remove impurities, the final double-stranded RNA targeting the citrus psyllid trehalase gene was obtained.

[0033] Example 6

[0034] Primers with restriction enzyme sites as shown in SEQ ID NO: 6 and SEQ ID NO: 7 were used to amplify the trehalase fragment targeting the citrus psyllid. The fragment was then inserted into the multiple cloning site of the L4440 plasmid using T4 DNA ligase to obtain a recombinant plasmid. The recombinant plasmid was transformed into competent HT115(DE3) Escherichia coli cells. The transformed strain was cultured in a medium at 38°C and 230 rpm. When the absorbance of the bacterial culture reached 0.5, isopropyl-β-D-thiogalactoside was added to a final concentration of 0.9 mM. The fermentation temperature was lowered to 29°C, and fermentation was induced for 10 hours. The bacterial pellet was collected and subjected to repeated freeze-thaw cycles combined with heat pulse treatment to disrupt the bacterial cell wall and membrane structure, releasing the intracellularly synthesized double-stranded RNA targeting the citrus psyllid trehalase gene. After centrifugation at 12500 rpm to remove impurities, the double-stranded RNA targeting the citrus psyllid trehalase gene was finally obtained.

[0035] Example 7

[0036] Primers with restriction enzyme sites as shown in SEQ ID NO: 6 and SEQ ID NO: 7 were used to amplify the trehalase fragment targeting the citrus psyllid. The fragment was then inserted into the multiple cloning site of the L4440 plasmid using T4 DNA ligase to obtain a recombinant plasmid. The recombinant plasmid was transformed into competent HT115(DE3) Escherichia coli cells. The transformed strain was cultured in a medium at 40°C and 240 rpm. When the absorbance of the bacterial culture reached 0.6, isopropyl-β-D-thiogalactoside was added to a final concentration of 1.0 mM. The fermentation temperature was lowered to 30°C, and fermentation was induced for 12 hours. The bacterial pellet was collected and subjected to repeated freeze-thaw cycles combined with heat pulse treatment to disrupt the bacterial cell wall and membrane structure, releasing the intracellularly synthesized double-stranded RNA targeting the citrus psyllid trehalase gene. After centrifugation at 13000 rpm to remove impurities, the double-stranded RNA targeting the citrus psyllid trehalase gene was finally obtained.

[0037] Example 8

[0038] The double-stranded RNA solution obtained in Example 2 was mixed with the modified graphene oxide nanocarrier solution at a volume ratio of 1:2. The mixture was stirred at 100 rpm for 30 min at 20 °C to construct a primary complex through electrostatic self-assembly. The resulting primary complex was then transferred to a high-frequency energy processing chamber and treated for 15 min under an acoustic field with a frequency of 40 kHz and a power density of 1.8 W / cm². This process eliminated the non-covalent aggregation between the double-stranded RNA, the polymer auxiliary components, and the nanosheets through acoustic cavitation. The treated complex was then pumped into an insecticide solution at a volume ratio of 1:10 and circulated and sheared at 3000 rpm for 10 min to allow the insecticide molecules to form a molecular-level dispersed distribution on the surface of the complex. The mixture was then cooled to room temperature to obtain the final product.

[0039] Example 9

[0040] The double-stranded RNA solution obtained in Example 3 was mixed with the modified graphene oxide nanocarrier solution at a volume ratio of 1:2. The mixture was stirred at 150 rpm for 40 min at 22 °C to construct a primary complex through electrostatic self-assembly. The resulting primary complex was then transferred to a high-frequency energy processing chamber and treated for 17 min under an acoustic field with a frequency of 50 kHz and a power density of 2.1 W / cm². This process eliminated the non-covalent aggregation between the double-stranded RNA, the polymer auxiliary components, and the nanosheets through acoustic cavitation. The treated complex was then pumped into an insecticide solution at a volume ratio of 1:12 and circulated and sheared at 4000 rpm for 15 min to allow the insecticide molecules to form a molecular-level dispersed distribution on the surface of the complex. The mixture was then cooled to room temperature to obtain the final product.

[0041] Example 10

[0042] The double-stranded RNA solution obtained in Example 4 was mixed with the modified graphene oxide nanocarrier solution at a volume ratio of 1:3. The mixture was stirred at 200 rpm for 50 min at 25 °C to construct a primary complex through electrostatic self-assembly. The resulting primary complex was then transferred to a high-frequency energy processing chamber and treated for 20 min under an acoustic field with a frequency of 60 kHz and a power density of 2.5 W / cm². This process eliminated the non-covalent aggregation between the double-stranded RNA, the polymer auxiliary components, and the nanosheets through acoustic cavitation. The treated complex was then pumped into an insecticide solution at a volume ratio of 1:15 and circulated and sheared at 5000 rpm for 20 min to allow the insecticide molecules to form a molecular-level dispersed distribution on the surface of the complex. The mixture was then cooled to room temperature to obtain the final product.

[0043] Example 11

[0044] The double-stranded RNA solution obtained in Example 5 was mixed with the modified graphene oxide nanocarrier solution at a volume ratio of 1:2. The mixture was stirred at 100 rpm for 30 min at 20 °C to construct a primary complex through electrostatic self-assembly. The resulting primary complex was then transferred to a high-frequency energy processing chamber and treated for 15 min under an acoustic field with a frequency of 40 kHz and a power density of 1.8 W / cm². This process eliminated the non-covalent aggregation between the double-stranded RNA, the polymer auxiliary components, and the nanosheets through acoustic cavitation. The treated complex was then pumped into an insecticide solution at a volume ratio of 1:10 and circulated and sheared at 3000 rpm for 10 min to allow the insecticide molecules to form a molecular-level dispersed distribution on the surface of the complex. The mixture was then cooled to room temperature to obtain the final product.

[0045] Example 12

[0046] The double-stranded RNA solution obtained in Example 6 was mixed with the modified graphene oxide nanocarrier solution at a volume ratio of 1:2. The mixture was stirred at 150 rpm for 40 min at 22 °C to construct a primary complex through electrostatic self-assembly. The resulting primary complex was then transferred to a high-frequency energy processing chamber and treated for 17 min under an acoustic field with a frequency of 50 kHz and a power density of 2.1 W / cm². This process eliminated the non-covalent aggregation between the double-stranded RNA, the polymer auxiliary components, and the nanosheets through acoustic cavitation. The treated complex was then pumped into an insecticide solution at a volume ratio of 1:12 and circulated and sheared at 4000 rpm for 15 min to allow the insecticide molecules to form a molecular-level dispersed distribution on the surface of the complex. The mixture was then cooled to room temperature to obtain the final product.

[0047] Example 13

[0048] The double-stranded RNA solution obtained in Example 7 was mixed with the modified graphene oxide nanocarrier solution at a volume ratio of 1:3. The mixture was stirred at 200 rpm for 50 min at 25 °C to construct a primary complex through electrostatic self-assembly. The resulting primary complex was then transferred to a high-frequency energy processing chamber and treated for 20 min under an acoustic field with a frequency of 60 kHz and a power density of 2.5 W / cm². This process eliminated the non-covalent aggregation between the double-stranded RNA, the polymer auxiliary components, and the nanosheets through acoustic cavitation. The treated complex was then pumped into an insecticide solution at a volume ratio of 1:15 and circulated and sheared at 5000 rpm for 20 min to allow the insecticide molecules to form a molecular-level dispersed distribution on the surface of the complex. The mixture was then cooled to room temperature to obtain the final product.

Claims

1. A trehalase gene double-stranded RNA nanoparticle complex for controlling citrus psyllids, characterized in that, By weight, the nanoparticle complex comprises the following components: 100-120 parts insecticide, 8-12 parts modified graphene oxide nanocarrier, and 4-6 parts double-stranded RNA targeting the trehalase gene of the citrus psyllid; wherein the double-stranded RNA targeting the trehalase gene of the citrus psyllid is prepared by either pathway A or pathway B: Path A: S1 Collect adult citrus psyllids and put them into an electric grinder. Add liquid nitrogen and grind at 1500-1600 rpm until a grayish-white powder with no visible tissue is formed. Add acidic lysis buffer to the powder and let it stand at 15-25℃ for 5-10 min to destroy the cell membrane structure and inactivate endogenous ribonuclease by using the liquid salt. Add 0.2-0.3 times the volume of chloroform to the lysis system, shake at 4000-5000 rpm for 10-15 s, and then centrifuge at 4-6℃ and 12000-13000 rpm for 15-20 min. The colorless and transparent upper aqueous phase was extracted and transferred to a nuclease-free centrifuge tube. An equal volume of isopropanol was added, and the mixture was incubated at -20 to -18°C for 30 to 40 minutes to drive the dehydration and precipitation of RNA molecules. After centrifugation at 12,000 to 13,000 rpm for 10 to 20 minutes, the supernatant was discarded. The precipitate was washed twice with ethanol solution to remove residual salts and organic solvents. The RNA precipitate was dissolved in 10 to 20 μL of ultrapure nuclease-free water to obtain a high-abundance total RNA matrix. S2. Take total RNA, add deoxyribonuclease I, and treat at 37-39℃ for 10-20 min to completely eliminate interference from genomic DNA; add oligodeoxythymidine primers, heat at 65-75℃ for 5-10 min to expand the secondary structure of RNA, and then quickly place on ice to cool; add reverse transcriptase, deoxyribonucleoside triphosphate mixture and reaction buffer, extend at 42-44℃ for 60-120 min, and then heat at 70-80℃ for 15-25 min to inactivate reverse transcriptase, finally obtaining full-length single-stranded cDNA containing the trehalase gene of citrus psyllid; S3 uses the primer pair shown in SEQ ID NO: 2 and SEQ ID NO:

3. Using the obtained cDNA as a template, DNA polymerase is added. The mixture is pre-denatured at 95-96℃ for 3-4 min; denatured at 98-100℃ for 10-20 s; annealed at 58-60℃ for 20-30 s; extended at 72-74℃ for 30-60 s; and cycled for a total of 35-40 times. Finally, it is fully extended at 70-72℃ for 5-10 min. The amplified product is subjected to agarose gel electrophoresis, and the target fragment is obtained using gel extraction technology. This fragment is used as template DNA for in vitro transcription and synthesis of double-stranded RNA targeting the trehalase gene. In step S4, the obtained template DNA, ATP, CTP, GTP, UTP, T7 RNA polymerase, and ribonuclease inhibitor were added to a centrifuge tube. The reaction system was incubated in a constant temperature dry bath at 37–40°C for 8–12 hours. After the reaction, the system was heated to 75–80°C and held for 5–10 minutes to eliminate non-specific pairing. The system was then cooled to 20–25°C at a forced cooling rate of 1.5–2°C per minute. During this process, single-stranded RNA molecules achieve precise base pairing through intermolecular hydrogen bonds, resulting in double-stranded RNA molecules with a specific spatial conformation targeting the trehalase gene. Path B: S5 used primers with restriction enzyme sites shown in SEQ ID NO: 6 and SEQ ID NO: 7 to amplify the trehalase fragment targeting the citrus psyllid. The fragment was then inserted into the multiple cloning site of the L4440 plasmid using T4 DNA ligase to obtain a recombinant plasmid. The recombinant plasmid was transformed into competent HT115(DE3) *E. coli* cells. The transformed strain was cultured in a medium at 37–40°C and 220–240 rpm. When the absorbance of the bacterial culture reached 0.4–0.6, isopropyl-β-D-thiogalactoside was added to a final concentration of 0.8–1.0 mM. The fermentation temperature was lowered to 28–30°C, and fermentation was induced for 8–12 hours. The bacterial pellet was collected and subjected to repeated freeze-thaw cycles combined with heat pulses to disrupt the bacterial cell wall and membrane structure, releasing the intracellularly synthesized double-stranded RNA targeting the citrus psyllid trehalase gene. The pellet was then subjected to 12,000–13,000 [units of temperature missing]. After centrifugation at rpm to remove impurities, the double-stranded RNA targeting the trehalase gene of the citrus psyllid was finally obtained.

2. The trehalase gene double-stranded RNA nanoparticle complex for controlling citrus psyllids as described in claim 1, characterized in that, The insecticide is one or more of imidacloprid, thiamethoxam, acetamiprid, and etoxazole in combination.

3. The trehalase gene double-stranded RNA nanoparticle complex for controlling citrus psyllids as described in claim 1, characterized in that, The acidic lysis buffer described in S1 is one or more of guanidine isothiocyanate, sodium dodecyl sarcosinate, and phenol in combination.

4. The trehalase gene double-stranded RNA nanoparticle complex for controlling citrus psyllids as described in claim 1, characterized in that, The DNA polymerase is one or more of Pfu DNA polymerase, KOD DNA polymerase, and Taq DNA polymerase.

5. The trehalase gene double-stranded RNA nanoparticle complex for controlling citrus psyllids as described in claim 1, characterized in that, The ribonuclease inhibitor is one or more of the following: recombinant placental ribonuclease inhibitor, diethyl pyrocarbonate, vanadium ribonucleoside complex, guanidine isothiocyanate, and sodium dodecyl sarcosinate.

6. The trehalase gene double-stranded RNA nanoparticle complex for controlling citrus psyllids as described in claim 1, characterized in that, The modified graphene oxide nanocarrier is a ternary topological nanosheet constructed by covalently grafting carboxylated graphene oxide with polyethylene glycol and polyethyleneimine. Its surface carries a positive charge and has a steric hindrance protection layer.

7. The trehalase gene double-stranded RNA nanoparticle complex for controlling citrus psyllids as described in claim 1, characterized in that, The preparation process of the trehalase gene double-stranded RNA nanoparticle complex for controlling citrus psyllids includes the following steps: S6 mixes double-stranded RNA solution with modified graphene oxide nanocarrier solution at a volume ratio of 1:2~3, and stirs at 100~200 rpm for 30~50 min at 20~25℃ to construct primary complex through electrostatic self-assembly. S7 The obtained primary complex was transferred to a high-frequency energy processing chamber and treated for 15-20 min under an acoustic field with a frequency of 40-60 kHz and a power density of 1.8-2.5 W / cm². The non-covalent aggregation between double-stranded RNA, polymer auxiliary components and nanosheets was eliminated through acoustic cavitation effect. S8 The treated complex is pumped into the insecticide solution at a volume ratio of 1:10~15, and circulated and cut at a speed of 3000~5000 rpm for 10~20 min to make the insecticide molecules form a molecular-level dispersed distribution on the surface of the complex. The mixture is then cooled to room temperature to obtain the final product.

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