Target fusion gene of nilaparvata lugens n1chs-cht1 and nanocarrier co-delivery system with lethal effect

Abstract

The application discloses a target fusion gene of brown planthopper NlCHS Cht1 and a nano co-delivery system with lethal effect; the nucleotide sequence is shown as SEQ ID NO:1. The nano pesticide comprises 1 part of zinc nitrate hexahydrate, 13-14 parts of 2-methylimidazole, 0.03-0.08 parts of buprofezin and 0.05-0.2 parts of ds NlCHS- Cht1 The nano co-delivery system can improve the insecticidal effect by combining RNAi with buprofezin, and can improve the sensitivity of the brown planthopper to buprofezin by interfering with the target gene, which provides a new idea and perspective for the resistance management of buprofezin, helps the application of buprofezin in pest control, improves the application effect of the RNAi technology in the brown planthopper control, and clearly defines the application potential of the combination of RNAi and chemical pesticides.​

Description

Technical Field

[0001] This invention relates to the field of pest control, specifically to a target fusion gene for brown planthoppers. NlCHS - Cht1 And its lethal nano-co-delivery system. Background Technology

[0002] Rice is my country's largest grain crop, and the losses caused by pests, diseases, and weeds every year are enormous. The brown planthopper is one of the most serious pests affecting rice production. Brown planthoppers have long-distance migratory habits, causing widespread damage and frequent outbreaks, resulting in severe economic losses. For a long time, chemical control has been the primary method for controlling brown planthoppers. However, due to the long-term unscientific and irrational use of chemical insecticides, the evolution of pesticide resistance in brown planthoppers has accelerated, leading to varying degrees of resistance to 33 insecticides, including thiamethoxam. The development of pesticide resistance in brown planthoppers reduces the control efficacy of many existing chemical insecticides. However, increasing the dosage of pesticides can cause a series of problems such as environmental pollution and agricultural product safety issues. Furthermore, the development of new chemical insecticides is extremely costly and time-consuming. Therefore, there is an urgent need to develop new methods for controlling brown planthoppers.

[0003] Suppressing key genes in pest growth and development using RNAi technology has become a hot research topic in pest control in recent years. RNAi-mediated gene silencing has advantages such as high specificity and efficiency. Suppressing key genes in pest growth and development using RNAi technology can hinder normal growth and development, and even lead to death. Therefore, RNAi technology has enormous application potential in pest control. Pest control based on RNAi can be achieved mainly through transgenic and non-transgenic strategies. However, the regulatory guidelines for transgenic organisms are still unclear, and their safety remains highly controversial, limiting their development. Non-transgenic strategies, which use exogenous dsRNA to control pests, currently face many problems limiting RNAi efficiency in practical applications. First, the lethal effect of the same target gene varies among different insects, increasing the difficulty of RNAi target gene selection. Second, dsRNA is unstable during delivery and prone to degradation, resulting in poor RNAi efficiency, and the delivery dose is often too small to induce a lethal effect. When using RNAi technology for pest control, many problems remain to be solved regarding the selection of target genes within the pest and improving RNAi efficiency.

[0004] Nanomaterials refer to particles with sizes between 1 and 100 nm. They possess characteristics such as small size, large specific surface area, and ease of modification, and are widely used in medicine for the delivery of drug molecules and biomolecules. In recent years, to improve the stability of dsRNA and the uptake efficiency of cells, nanomaterials have also been increasingly used for the delivery of insect dsRNA. Many studies have demonstrated that using nanomaterials to encapsulate and deliver dsRNA can improve its interference efficiency. However, in practical applications, it is not only necessary to consider the delivery performance of the carrier, but also factors such as environmental risks and economic benefits. For example, the raw materials for synthesizing liposomes, such as phospholipids, are expensive; cationic polymers are difficult to degrade and exhibit significant cytotoxicity; and nanomicelles still have problems in transmembrane absorption. Further selection of suitable nanomaterial delivery carriers is needed. Metal-organic frameworks (MOFs) are a class of porous materials self-assembled from metal ions and organic ligands. They possess unique advantages such as high porosity, large specific surface area, and structural diversity. Their porous structure and multiple organic-inorganic interaction sites make them easy to bind to various biomolecules (such as proteins and nucleic acids), making them a potential nucleic acid carrier. Zinc ions are an endogenous metal ion, formed by the metal ion Zn. 2+ The metal-organic framework formed with imidazole or its derivatives is the zeolite imidazole framework (ZIF), which exhibits good biocompatibility. The most representative ZIF is ZIF-8, which is composed of Zn... 2+ ZIF-8, formed by coordination with dimethylimidazole, not only possesses the advantages of simple synthesis of metal-organic framework structures, easy surface modification, large specific surface area, tunable pore size, and strong drug loading capacity, but also has the characteristics of inexpensive raw materials, good biocompatibility, and pH responsiveness, making it a promising candidate for dsRNA delivery.

[0005] RNAi technology, when combined with existing insecticides, can further enhance insecticidal efficacy. RNAi technology disrupts the normal growth and development of insects by silencing target genes, assisting insecticides in overcoming the barriers of the insect's body wall or periphyte, thus enhancing their effectiveness. Buprofezin, a chitin synthesis inhibitor, inhibits insect molting, affecting insect growth and development, leading to pest mortality. It is highly toxic to many hemiptera pests and less toxic to non-target mammals, and is widely used for the control of brown planthoppers. However, since 2013, brown planthoppers have developed high levels of resistance to buprofezin, and in 2014, the National Agricultural Technology Extension Service Center recommended suspending its use in brown planthopper control. Studies have shown that mutations in chitin synthase are the cause of resistance in brown planthoppers. Interfering with chitin synthase expression can increase the sensitivity of brown planthoppers to buprofezin. Therefore, the combined use of RNAi technology and buprofezin can be used for the control of brown planthoppers.

[0006] Current methods for controlling brown planthoppers have the following problems: 1) Agricultural pests are becoming more resistant to pesticides, and the effectiveness of chemical pesticides is decreasing, so there is an urgent need to develop new control methods; 2) The problem of pesticide resistance needs to be addressed; 3) RNAi technology can be used for pest control, but the efficiency of in vitro delivery of dsRNA is relatively low; 4) Nanomaterials can be used to deliver dsRNA, but existing vectors have problems such as high synthesis cost and high cytotoxicity. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a brown planthopper target fusion gene. NlCHS - Cht1 This invention utilizes a lethal nano-co-delivery system based on two key genes in the chitin synthesis and degradation pathway of the brown planthopper—chitin synthase (Chitin synthase). NlCHS ) and chitinase 1 ( NlCht1 The target gene is used as the target gene, and the fusion is used to obtain the target fusion gene. NlCHS - Cht1 Based on biotechnology, ds with high lethality were prepared. NlCHS-Cht1 Further, based on the ZIF-8 nanocarrier, ds NlCHS-Cht1 Co-delivery system with pesticide molecule thiamethoxam (Buprofezin@ds) NlCHS-Cht1 @ZIF-8), which is the lethal nano-co-delivery system Buprofezin@ds NlCHS-Cht1 @ZIF-8, this nano-co-delivery system can enhance insecticidal efficacy through the combined use of RNAi and thiamethoxam, and can also increase the sensitivity of brown planthoppers to thiamethoxam by interfering with target genes. This provides a new approach and perspective for the management of thiamethoxam resistance, which is conducive to the application of thiamethoxam in pest control. At the same time, it improves the application effect of RNAi technology in the control of brown planthoppers, clarifies the application potential of RNAi combined with chemical pesticides, and provides a new strategy for the precise control of brown planthoppers.

[0008] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a target fusion gene for brown planthoppers. NlCHS - Cht1 Its nucleotide sequence is shown in SEQ ID NO: 1.

[0009] The present invention also provides a recombinant vector pESI-T- NlCHS - Cht1 The recombinant vector contains the aforementioned brown planthopper target fusion gene. NlCHS - Cht1The expression vector is a pESI-T vector.

[0010] The present invention also provides a recombinant vector L4440- NlCHS-Cht1 The recombinant vector contains the aforementioned brown planthopper cytochrome P450 gene. NlCYP6ER1 The expression vector is L4440.

[0011] The present invention also provides a recombinant vector L4440- containing the above-mentioned recombinant vector. NlCHS-Cht1 The host cell, wherein the host cell is an HT115 competent cell.

[0012] The present invention also provides a method for extracting ds using the above-mentioned host cells. NlCHS-Cht1 The method, said method, involves placing a substance containing the recombinant vector L4440- NlCHS-Cht1 Host cell culture was adjusted to approximately OD600 = 0.40, and IPTG was added to a final concentration of 1 mM for induction of expression. The cells were then cultured at 37°C with shaking for 4 hours. Total bacterial RNA was extracted, and the purified dsRNA was obtained, which is the dsRNA. NlCHS-Cht1.

[0013] This invention also provides a lethal nano-co-delivery system, wherein the raw materials of the nano-insecticide, by weight, include 1 part of zinc nitrate hexahydrate, 13-14 parts of 2-methylimidazole, 0.03-0.08 parts of thiamethoxam, and 0.05-0.2 parts of ds prepared by the above method. NlCHS-Cht1 .

[0014] Furthermore, the raw materials of the nano-sized insecticide, by weight ratio, include 1 part of zinc nitrate hexahydrate, 13.86 parts of 2-methylimidazole, 0.06 parts of thiamethoxam, and 0.10 parts of ds NlCHS-Cht1 .

[0015] This invention also provides a method for preparing a lethal nano-co-delivery system, comprising the following steps: 1) Weigh out 1 part by weight of zinc nitrate hexahydrate solution, 13-14 parts by weight of 2-methylimidazole solution, 0.03-0.08 parts by weight of thiamethoxam, and 0.05-0.2 parts by weight of the ds prepared by the above method. NlCHS-Cht1 ; 2) Dissolve zinc nitrate hexahydrate and 2-methylimidazole in 0.1% Triton X-100 aqueous solution to obtain zinc nitrate hexahydrate Triton X-100 aqueous solution and 2-methylimidazole Triton X-100 aqueous solution, respectively; 3) Dissolve the thiamethoxam solution in dimethyl sulfoxide (DMSO) to obtain a saturated thiamethoxam DMSO solution; 4) ds NlCHS-Cht1 Add an aqueous solution of zinc nitrate hexahydrate (Triton X-100) and stir at room temperature to obtain a reaction solution. Then, add a saturated DMSO solution of thiamethoxam to the reaction solution and stir to mix thoroughly at room temperature. Finally, add an aqueous solution of 2-methylimidazolium (Triton X-100) and react at room temperature. After the reaction is complete, collect the nanoparticles by centrifugation, wash three times with deionized water and anhydrous ethanol, and dry under vacuum. The final product is the lethal nano-co-delivery system Buprofezin@ds. NlCHS- Cht1 @ZIF-8.

[0016] Furthermore, in step 4), the centrifugation speed is 10,000 rpm.

[0017] The present invention also provides an application of the above-mentioned nano-co-delivery system in the control of brown planthoppers.

[0018] The beneficial effects of this invention are: (1) This invention utilizes the HT115-L4440 system to induce expression of dsRNA, which greatly reduces the synthesis cost of dsRNA; (2) This invention utilizes ZIF-8 nanomaterials to deliver dsRNA, thereby improving the silencing efficiency of target genes in brown planthoppers; (3) The present invention constructs a co-loading system of insecticide and dsRNA, and uses ZIF-8 to simultaneously load dsRNA and insecticide. The synthesis method is simple and the reaction conditions are mild, and it has the potential for practical application. (4) The present invention combines RNAi with thiamethoxam to improve the insecticidal effect and can also increase the sensitivity of brown planthopper to thiamethoxam by interfering with target genes, which is helpful for the resistance management of thiamethoxam and the application of RNAi technology. (5) By changing the target gene and the insecticide, the system can also be used to control the resistance of other pests.

[0019] In summary, the nano-co-delivery system of this invention can enhance the insecticidal effect by combining RNAi with thiamethoxam and increase the sensitivity of brown planthoppers to thiamethoxam by interfering with target genes. This provides a new approach and perspective for the management of thiamethoxam resistance, which is conducive to the application of thiamethoxam in pest control. At the same time, it improves the application effect of RNAi technology in the control of brown planthoppers, clarifies the application potential of RNAi combined with chemical pesticides, and provides a new strategy for the precise control of brown planthoppers. Attached Figure Description

[0020] Figure 1 ds induced expression of purified E. coli NlCHS-Cht1 Gel electrophoresis image (Note: Marker: 2,000bp); Figure 2 For Buprofezin@ds NlCHS-Cht1 Transmission electron microscope image of @ZIF-8 (scale bar = 100 nm). Figure 3 For Buprofezin@ds NlCHS-Cht1 Scanning electron microscope image of @ZIF-8 (scale bar = 500 nm); Figure 4 For Buprofezin@ds NlCHS-Cht1 Particle size distribution diagram of @ZIF-8; Figure 5 For Buprofezin@ds NlCHS-Cht1 Element distribution diagram of @ZIF-8; Figure 6 for ZIF-8, Buprofezin@ZIF-8, ds NlCHS-Cht1 @ZIF-8 and Buprofezin@ds NlCHS- Cht1 XRD pattern of @ZIF-8; Figure 7 for Buprofezin, ZIF-8, Buprofezin@ZIF-8, ds NlCHS-Cht1 @ZIF-8 and Buprofezin@ds NlCHS-Cht1 Infrared spectrum of @ZIF-8; Figure 8 for ZIF-8, Buprofezin@ZIF-8, ds NlCHS-Cht1 @ZIF-8 and Buprofezin@ds NlCHS- Cht1 Zeta potential values ​​of @ZIF-8; Figure 9 A graph showing the relative expression of genes in brown planthoppers under different treatment groups; In the figure, the bars represent the mean ± standard error, and different letters on the bars indicate significant differences (P < 0.05). Figure 10 Buprofezin@ds at a concentration of 8 mg / L NlCHS-Cht1 Survival curves of brown planthoppers under different treatments using @ZIF-8 (statistical analysis was performed using the log-rank (Mantel-Cox) test); in the figure, The significance level is set to P < 0.05); Figure 11 Buprofezin@ds in a semi-field setting NlCHS-Cht1 Image showing the effect of @ZIF-8 on brown planthopper control. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0022] Example 1 Fusion Gene NlCHS-Cht1 Build Total RNA was extracted from brown planthoppers according to the instructions of TaKaRa's RNAiso Plus total RNA extraction reagent for plant and animal tissues. First-strand cDNA synthesis was performed using the Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigester plus) reverse transcription kit (YEASEN). [The remaining text appears to be incomplete and requires further context.] NlCHS and NlCht1 Primers for amplifying a 250 bp fragment were designed in the gene coding region (sequences are shown in Table 1). Using cDNA as a template, the fragment was cloned using RT-PCR. NlCHS and NlCht1 Sequence fragments and PCR products were detected by agarose gel electrophoresis. The gel was recovered according to the EZNA gel recovery kit instructions. NlCHS and NlCht1 Gene fragments were extracted, and the recovered PCR products were subjected to fusion PCR. Following the reaction, agarose gel electrophoresis was performed to detect the fragments, and fragments of 500 bp were recovered. Hieff Clone from Takara was used. ® The Zero TOPO-TA Simple Cloning Kit ligated the recovered fragment to the pESI-T vector. The ligation product was transformed into Trelief™ 5α competent cells and plated on solid LB agar plates containing ampicillin. After incubation for approximately 12 hours, single white colonies were picked and cultured in LB + Amp liquid medium. Preliminary validation was performed using 2×Hieff PCR Mester Mix (YEASEN). Samples with verified results were further sequenced to obtain a fusion gene fragment of 500 bp. NlCHS-Cht1 .

[0023] Table 1 Primer sequences for fusion gene fragments Example 2 Fusion Gene NlCHS-Cht1 dsRNA synthesis 1. Recombinant vector L4440- NlCHS-Cht1 Build L4440 and pESI-T- respectively NlCHS-Cht1The plasmid was digested with XbaⅠ and XhoⅠ (TaKaRa) restriction endonucleases, and the target band of the correct size was recovered using the EZNA gel extraction kit. The recovered fragment was ligated, and the ligation product was transformed into Trelief TM5α competent cells. The cells were plated on solid LB agar plates containing ampicillin and incubated for approximately 12 h. Single white colonies were picked and cultured in LB + Amp liquid medium. Colony PCR was performed using 2×Hieff PCR Mester Mix (YEASEN). The plasmid was extracted using the OMEGA EZNA Plasmid Mini Kit, which yielded the recombinant vector L4440-. NlCHS-Cht1 .

[0024] 2. Containing the above-mentioned recombinant vector L4440- NlCHS-Cht1 host cells The recombinant vector L4440- will be constructed. NlCHS-Cht1 After transformation into HT115 competent cells, the cells were plated on solid LB agar plates containing ampicillin and cultured for approximately 12 hours. White single colonies were then picked and cultured in LB + Amp liquid medium. Preliminary validation of the bacterial culture was performed using 2×Hieff PCR Mester Mix (YEASEN). Positive bacterial cultures were selected for sequencing and the cultures were cryopreserved.

[0025] 3. Extract ds NlCHS-Cht1 Method The recombinant vector L4440- was cultured in liquid LB medium at 37°C with shaking. NlCHS-Cht1 HT115 strain, to OD 600 Add approximately 0.40 g of IPTG to a final concentration of 1 mM for induction of expression, and continue incubation at 37°C with shaking for 4 hours. Extract total bacterial RNA, and purify the resulting dsRNA (ds NlCHS-Cht1 The concentration and quality of dsRNA were detected by spectrophotometer and 1% agarose gel electrophoresis. Figure 1 Store at -80℃ for later use.

[0026] Depend on Figure 1 It can be seen that a bright band exists at around 500 bp, with the same size as the target band, indicating that DS... NlCHS- Cht1 It was successfully prepared.

[0027] Example 3 A method for preparing a lethal nano-co-delivery system includes the following steps: 1) Weigh 0.5 g of zinc nitrate hexahydrate solution, 6.928 g of 2-methylimidazole solution, 30 mg of thiamethoxam, and 50 mg of the ds prepared by the above method. NlCHS-Cht1 ; 2) Dissolve zinc nitrate hexahydrate and 2-methylimidazole in 0.1% Triton X-100 aqueous solution to obtain zinc nitrate hexahydrate Triton X-100 aqueous solution and 2-methylimidazole Triton X-100 aqueous solution, respectively; 3) Dissolve the thiamethoxam solution in dimethyl sulfoxide (DMSO) to obtain a saturated thiamethoxam DMSO solution; 4) ds NlCHS-Cht1 Add an aqueous solution of zinc nitrate hexahydrate (Triton X-100) and stir at room temperature to obtain a reaction solution. Then, add a saturated DMSO solution of thiamethoxam to the reaction solution and stir to mix thoroughly at room temperature. Finally, add an aqueous solution of 2-methylimidazolium (Triton X-100) and react at room temperature. After the reaction is complete, collect the nanoparticles by centrifugation at 10,000 rpm, wash three times with deionized water and anhydrous ethanol, and vacuum dry. Finally, obtain the lethal nano-co-delivery system Buprofezin@ds. NlCHS-Cht1 @ZIF-8.

[0028] For the above-mentioned nano-co-delivery system Buprofezin@ds NlCHS-Cht1 @ZIF-8 was used for scanning electron microscopy, transmission electron microscopy, particle size distribution analysis, and energy dispersive spectroscopy characterization. The results are as follows: Figure 2-5 As shown, the nano-co-delivery system Buprofezin@ds NlCHS-Cht1 The nanoparticles of @ZIF-8 have regular shapes and uniform particle sizes, with an average particle size of 105.33 nm. C, O, N, P, S, and Zn elements are all present in Buprofezin@ds NlCHS-Cht1 @ZIF-8 nanoparticles; for the nano-co-delivery system Buprofezin@ds NlCHS-Cht1 XRD, Fourier transform infrared spectroscopy, and Zeta potential analysis were performed on @ZIF-8, and the results are as follows: Figure 6-8 As shown, this further proves that ZIF-8 is effective against ds NlCHS-Cht1 Successful loading of thiamethoxam and thiamethoxam.

[0029] Example 4: Buprofezin@ds Nanoscale Co-delivery System NlCHS-Cht1 Indoor bioactivity assay of ZIF-8 against brown planthopper To determine Buprofezin@ds NlCHS-Cht1 @ZIF-8 inhibits chitin synthase in brown planthoppers ( NlCHS ) and chitinase 1 ( NlCht1The effect of ds on gene expression of brown planthopper was detected using qRT-PCR based on rice seedling soaking method. GFP ds NlCHS-Cht1 ds NlCHS-Cht1 @ZIF-8 and Buprofezin@ds NlCHS-Cht1 @ZIF-8 treatment of brown planthopper chitin synthase ( NlCHS ) and chitinase 1 ( NlCht1 The expression level of genes.

[0030] The results are as follows Figure 9 As shown: with ds GFP In comparison, ds NlCHS-Cht1 and ds NlCHS-Cht1 @ZIF-8 treatment for 24 h resulted in the synthesis of chitin synthase in brown planthoppers ( NlCHS ) and chitinase 1 ( NlCht1 The expression level of the gene did not change significantly, while in Buprofezin@d sNlCHS-Cht1 @ZIF-8 treatment for 24 h resulted in the synthesis of chitin synthase in brown planthoppers ( NlCHS ) and chitinase 1 ( NlCht1 Gene expression levels compared to ds GFP Expression levels were significantly downregulated in all treatment groups. This indicates that Buprofezin@ds NlCHS- Cht1 @ZIF-8 can significantly inhibit chitin synthase in brown planthoppers ( NlCHS ) and chitinase 1 ( NlCht1 Gene expression.

[0031] Based on this, using water and ZIF-8 treatment as controls, the levels of thiamethoxam, Buprofezin@ZIF-8, and Buprofezin@ds were determined using the rice seedling immersion method. NlCHS-Cht1 The toxicity of @ZIF-8 to 3rd instar brown planthoppers was investigated. Five different concentrations were set up for each treatment, with each concentration replicated five times. Each replicate was inoculated with 15 3rd instar brown planthopper nymphs and fed continuously for 5 days. Mortality rates of brown planthoppers in different treatment groups were measured, and the LC50 under different treatments was calculated. 50 value.

[0032] The results are shown in Table 2. After 5 days of treatment, Buprofezin@ds NlCHS-Cht1 @ZIF-8 LC for brown planthopper 50 The LC50 value of Buprofezin@ZIF-8 against brown planthopper was 2.24 mg / L. 50 The LC50 value of thiamethoxam technical grade against brown planthopper was 3.41 mg / L. 50 The value was 4.03 mg / L, Buprofezin@ds NlCHS-Cht1@ZIF-8 is significantly more toxic to brown planthoppers than thiamethoxam technical grade, with a synergistic effect ratio of 1.78. This indicates that Buprofezin@ds NlCHS-Cht1 @ZIF-8 exhibits good biological activity against brown planthoppers.

[0033] Table 2 Buprofezin@ds NlCHS-Cht1 @ZIF-8 Bioactivity against Brown Planthopper Note: a Synergistic ratio = LC of thiamethoxam 50 / LC of other processing groups 50 If the 95% confidence interval for the synergistic ratio does not include 1.0, it indicates that the other treatment groups have a significant synergistic effect on the toxicity of thiamethoxam.

[0034] like Figure 10 As shown: Compared to the thiamethoxam technical treatment group, brown planthoppers showed better control in Buprofezin@ds NlCHS-Cht1 The death rate was faster under @ZIF-8 treatment. The results indicate that ZIF-8 nanocarriers can simultaneously deliver thiamethoxam and ds... NlCHS- Cht1 It can significantly enhance the toxicity of thiamethoxam to brown planthoppers.

[0035] Example 5 Buprofezin@ds NlCHS-Cht1 @ZIF-8's protective effect on rice To further evaluate Buprofezin@ds NlCHS-Cht1 The protective effect of @ZIF-8 on rice in a semi-field setting was evaluated by spraying different treatment groups onto rice seedlings to control brown planthoppers. A thiamethoxam concentration of 4 mg / L was selected as the treatment concentration, and seven treatment groups were set up: water (Control), ZIF-8, and ds. NlCHS- Cht1 ,buprofezin,ds NlCHS-Cht1 @ZIF-8, Buprofezin@ZIF-8 and Buprofezin@ dsNlCHS-Cht1 @ZIF-8, different treatment solutions were sprayed onto the leaves and stems of potted rice. After air drying, 100 third-instar brown planthoppers were placed in every 30 rice seedlings for feeding. The rice seedlings were photographed 7 days after treatment to observe their growth.

[0036] like Figure 11 As shown: After 7 days of treatment, the water (Control), ZIF-8, and ds NlCHS-Cht1 ,buprofezin and ds NlCHS-Cht1The rice in the @ZIF-8 treatment group withered after being fed on by brown planthoppers, while the rice in the Buprofezin@ZIF-8 and Buprofezin@ds treatment groups... NlCHS-Cht1 The rice in the @ZIF-8 treatment group grew well, and compared to the Buprofezin@ZIF-8 treatment group, the Buprofezin@ds treatment group showed better growth. NlCHS-Cht1 The rice plants in the @ZIF-8 treatment group were taller. In a semi-field setting, Buprofezin@ds NlCHS-Cht1 @ZIF-8 provides good protection for rice.

[0037] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A lethal nano-co-delivery system, characterized in that: The raw materials of the nano-co-delivery system, by weight, include 1 part zinc nitrate hexahydrate, 13-14 parts 2-methylimidazole, 0.03-0.08 parts thiamethoxam, and 0.05-0.2 parts ds NlCHS-Cht1 ; where ds NlCHS-Cht1 The preparation method involves using recombinant vector L4440- NlCHS-Cht1 HT115 competent cells were cultured and shaken to OD. 600 Add 0.40 g of IPTG to a final concentration of 1 mM, induce expression, and culture at 37°C with shaking for 4 h. Extract total RNA from the bacteria, and purify the resulting dsRNA, which is the dsRNA. NlCHS-Cht1 ; wherein, the recombinant vector L4440- NlCHS-Cht1 It contains a fusion gene targeting the brown planthopper. NlCHS - Cht1 The expression vector, and the brown planthopper target fusion gene NlCHS - Cht1 The nucleotide sequence is shown in SEQ ID NO:

1.

2. The lethal nano-co-delivery system according to claim 1, characterized in that: The raw materials of the nano-co-delivery system, by weight, include 1 part zinc nitrate hexahydrate, 13.86 parts 2-methylimidazole, 0.06 parts thiamethoxam, and 0.10 parts ds NlCHS-Cht1 .

3. A method for preparing the lethal nano-co-delivery system according to claim 1, characterized in that: Includes the following steps: 1) Weigh out 1 part of zinc nitrate hexahydrate, 13-14 parts of 2-methylimidazole, 0.03-0.08 parts of thiamethoxam, and 0.05-0.2 parts of ds according to the following weight ratios: NlCHS-Cht1 ; 2) Dissolve zinc nitrate hexahydrate and 2-methylimidazole in 0.1% Triton X-100 aqueous solution to obtain zinc nitrate hexahydrate Triton X-100 aqueous solution and 2-methylimidazole Triton X-100 aqueous solution, respectively; 3) Dissolve the thiamethoxam solution in dimethyl sulfoxide (DMSO) to obtain a saturated thiamethoxam DMSO solution; 4) ds NlCHS-Cht1 Add an aqueous solution of zinc nitrate hexahydrate (Triton X-100) and stir at room temperature to obtain a reaction solution. Then, add a saturated DMSO solution of thiamethoxam to the reaction solution and stir to mix thoroughly at room temperature. Finally, add an aqueous solution of 2-methylimidazolium (Triton X-100) and react at room temperature. After the reaction is complete, collect the nanoparticles by centrifugation, wash three times with deionized water and anhydrous ethanol, and dry under vacuum. The final product is the lethal nano-co-delivery system Buprofezin@ds. NlCHS-Cht1 @ZIF-8.

4. The method for preparing the lethal nano-co-delivery system according to claim 3, characterized in that: In step 4), the centrifugation speed is 10,000 rpm.

5. The application of the nano-co-delivery system according to claim 1 in the control of brown planthoppers.

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