Brown planthopper gene NlCYP303A1 and nano nucleic acid pesticide thereof

Abstract

The invention discloses a brown planthopper gene NlCYP303A1 and a nano nucleic acid pesticide of the brown planthopper gene NlCYP303A1. The nucleotide sequence of the gene NlCYP303A1 disclosed by the invention is as shown in SEQ ID NO: 1; the nano nucleic acid pesticide is mainly prepared from zinc nitrate hexahydrate, dsN1CYP303A1 and 2-methylimidazole. The gene NlCYP303A1 closely related to the ecdysis process of the brown planthopper is identified, and silencing of the gene can cause ecdysis failure of the brown planthopper and reduce the survival rate of the brown planthopper; furthermore, a dsN1CYP303A1-coated ZIF-8 nano nucleic acid pesticide delivery system is constructed on the basis of a nano carrier ZIF-8, and the biological activity of the dsN1CYP303A1-coated ZIF-8 nano nucleic acid pesticide delivery system is measured; results show that the nano nucleic acid pesticide delivery system overcomes the defect that the service life of dsRNA is short, and the control effect of the nano nucleic acid pesticide delivery system on brown planthopper is remarkably improved. The potential application value of RNAi in pest control is improved, and a new strategy is provided for pest drug resistance treatment.

Description

Technical Field

[0001] The invention relates to the field of insect pest resistance mechanism and prevention and control, and in particular to a brown planthopper gene N1CYP303A1 and a nano nucleic acid pesticide thereof. Background Art

[0002] Agricultural pests pose a serious threat to global crop production. Chemical pesticides have made great contributions to pest control and food security. However, improper use of pesticides has led to the rapid development of resistance in many pests, rendering many existing pesticides ineffective. Therefore, there is an urgent need to develop new and efficient pest control technologies. In recent years, nucleic acid pesticides targeting pest lethal genes have been gradually promoted as an emerging technology. RNA interference (RNAi) is an evolutionarily ubiquitous gene regulation mechanism. Silencing key genes in pests through RNAi can lead to phenotypes such as death or decreased reproductive capacity, and has the potential for efficient pest control. Nano nucleic acid pesticides produced based on RNAi technology are characterized by high efficiency and precision, which are superior to traditional methods including chemical pesticides.

[0003] Identifying the best target genes is key to using RNAi technology for pest control. Previous studies have shown that silencing immune-related genes can lead to a decrease in the ability of insects to resist bacterial infection, thereby increasing the mortality of insects after bacterial infection. For example, knocking down the prophenoloxidase (PPO) gene significantly increased the infection rate of bacteria and fungi in pea aphids (Acyrthosiphon pisum), leading to an increase in their mortality. In addition, studies have shown that silencing key genes in the digestive and excretory systems of insects can significantly reduce their survival rates. For example, silencing the DEAD-box (DDX) genes necessary for maintaining the normal function of the digestive organs in locusts (Locusta migratoria) can lead to a lethal phenotype. Silencing the highly expressed ATP1A1 and AQP genes in the excretory organs of brown planthoppers (Nilaparvata lugens) can lead to obvious lethal phenotypes and greatly reduce the production of honeydew. In addition, genes involved in the insect molting process have received the most attention. Injection of dsRNA targeting the peroxidase gene (Pxd) into the last instar larvae of the rice leaf roller (Chilo suppressalis) disrupts the hardening of the cuticle during the metamorphosis from larva to pupa, ultimately leading to the death of the larvae and pupae. Halloween genes, including spook (CYP307A1), phantom (CYP306A1), disembodied (CYP302A1), shadow (CYP315A1), and shade (CYP314A1), are involved in the biosynthesis of the molting hormone 20-hydroxyecdysone (20E). Silencing these genes in Bemisia tabaci significantly increases their mortality. It can be seen that nucleic acid pesticides that target and regulate key genes in the molting and metamorphosis process have broad application prospects.

[0004] RNAi technology has shown great potential in pest control, but the poor persistence and stability of dsRNA have limited its application in pest control. At present, nanoparticle-mediated dsRNA delivery systems have shown advantages in overcoming this bottleneck. Nanocarrier-based gene delivery systems are widely used in the pharmaceutical field. For example, genes can reach targets through nanocarriers and regulate the expression of related genes, thereby enhancing the therapeutic effect of chemotherapeutic drugs. In agriculture, nanoparticles have been proven to be very effective in delivering dsRNA. For example, in order to improve the stability of dsRNA, block copolymers of polyethylene glycol-polylysine were designed to protect dsRNA from premature degradation, promote the persistence of dsRNA in the intestinal system, and thus achieve effective control of migratory locusts. Studies have found that silencing CsCYP15C1 can significantly increase the mortality of rice leaf rollers, and further encapsulating the dsRNA in DMAEMA-based polycationic nanomaterials can further increase the mortality of larvae. The above studies show that it is feasible to use nanocarrier-based gene delivery systems to deliver dsRNA to control pests.

[0005] Brown planthopper is one of the most important rice pests, causing serious damage to agricultural production every year. Brown planthopper is an insect with incomplete metamorphosis and needs to undergo multiple molting to complete development. Previous studies have shown that genes related to molting are very important in the growth and development of brown planthopper. Therefore, identifying genes that are critical to the molting process is crucial for controlling brown planthopper using RNAi technology, and using nanomaterials to encapsulate dsRNA to enhance its stability and persistence is a practical method for controlling brown planthopper.

[0006] At present, the co-delivery system that encapsulates dsRNA and pesticides at the same time has improved the control effect of pesticides to a certain extent, but it still has not solved the problem of pesticide application. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a brown planthopper gene NlCYP303A1 and a nano nucleic acid pesticide thereof. The present invention identifies the gene NlCYP303A1 that is closely related to the molting process of the brown planthopper, and silencing the gene can cause the brown planthopper to fail to molt and reduce its survival rate. The present invention protects dsNlCYP303A1 from degradation through ZIF-8, improves the delivery efficiency of dsNlCYP303A1, improves the application effect of RNAi technology in the control of brown planthoppers, provides a new strategy for the control of brown planthoppers, and provides a new direction and new thinking for the management of insecticide resistance.

[0008] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0009] The invention provides a brown planthopper gene N1CYP303A1, and the nucleotide sequence of the gene N1CYP303A1 is shown in SEQ ID NO:1.

[0010] The present invention also provides a primer pair for obtaining the brown planthopper gene N1CYP303A1, the primer pair is N1CYP303A1-F2 and N1CYP303A1R2, and the nucleotide sequences thereof are shown in SEQ ID NOs: 2 and 3, respectively.

[0011] The present invention also provides a recombinant vector L4440-NlCYP303A1, wherein the recombinant vector is an expression vector containing the gene NlCYP303A1 according to claim 1, wherein the expression vector is L4440.

[0012] The present invention also provides a host cell containing the recombinant vector L4440-N1CYP303A1, wherein the host cell is a HT115 competent cell.

[0013] The present invention also provides a method for inducing the extraction of dsNlCYP303A1 from the host cell, wherein the host cell containing the recombinant vector L4440-NlCYP303A1 is cultured to an OD of 600 =0.40, add IPTG to a final concentration of 1 mM, induce expression, culture at 37°C with shaking for 4 h, extract total bacterial RNA, and dsRNA obtained after purification is dsNlCYP303A1.

[0014] The present invention also provides an application of dsNlCYP303A1 prepared by the preparation method in preventing and controlling brown planthoppers.

[0015] The present invention also provides a nano nucleic acid pesticide. The raw materials of the nano nucleic acid pesticide include, by weight, 1 part of zinc nitrate hexahydrate, 13-15 parts of 2-methylimidazole and 0.06-0.1 parts of dsNlCYP303A1 prepared by the preparation method.

[0016] Furthermore, the raw materials of the nano-nucleic acid pesticide include, by weight, 1 part of zinc nitrate hexahydrate, 13.86 parts of 2-methylimidazole and 0.08 parts of dsNlCYP303A1.

[0017] The present invention also provides a method for preparing a nano nucleic acid pesticide, comprising the following steps:

[0018] (1) Weigh, by weight, 1 part of zinc nitrate hexahydrate, 13 to 15 parts of 2-methylimidazole, and 0.06 to 0.1 parts of dsNlCYP303A1 prepared by the preparation method;

[0019] (2) dissolving zinc nitrate hexahydrate and 2-methylimidazole in water to obtain zinc nitrate hexahydrate solution and 2-methylimidazole solution respectively;

[0020] (3) dissolving dsNlCYP303A1 in a zinc nitrate hexahydrate solution, mixing with a 2-methylimidazole solution, and stirring at room temperature to obtain a mixed solution;

[0021] (4) The mixed solution was centrifuged, the waste liquid was discarded, anhydrous ethanol was added to wash the precipitate three times, the precipitate was collected, and vacuum dried to obtain the nano-nucleic acid pesticide.

[0022] The present invention also provides an application of the nano nucleic acid pesticide in preventing and controlling brown planthoppers.

[0023] Beneficial effects of the present invention:

[0024] 1. The gene NlCYP303A1 and the nano nucleic acid pesticide thereof of the present invention can significantly inhibit the molting behavior of brown planthoppers, thereby achieving a highly effective killing effect, providing a new idea for the prevention and control of brown planthoppers, helping to reduce the application of chemical pesticides in pest control, and at the same time promoting the popularization of RNAi technology in pest control, providing a new strategy for achieving efficient and accurate prevention and control of brown planthoppers.

[0025] 2. The dsNlCYP303A1@ZIF-8 nano nucleic acid pesticide preparation method of the present invention can improve the delivery efficiency of dsNlCYP303A1, solve the problem of poor persistence and stability of dsRNA, and thus improve the field control efficiency.

[0026] 3. The HT115-L4440 system is used to induce expression to obtain dsRNA, which greatly reduces the cost of dsRNA synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the NlCYP303A1 gene cloning and sequence analysis diagram;

[0028] In the figure, A is the genomic structure of the NlCYP303A1 gene;

[0029] B is the alignment of the full-length amino acid sequences of CYP303A1 from different insects;

[0030] C is the phylogenetic analysis diagram of CYP303A1 gene;

[0031] Figure 2 This is the result of gel electrophoresis detection of dsNlCYP303A1 induced expression;

[0032] Figure 3 This is a diagram of the characterization results of dsNlCYP303A1@ZIF-8;

[0033] In the figure, A and C are transmission electron microscopy and scanning electron microscopy images of ZIF-8, respectively;

[0034] B and D are transmission electron microscopy and scanning electron microscopy images of dsNlCYP303A1@ZIF-8, respectively;

[0035] E and F are the particle size distribution diagrams of ZIF-8 and dsNlCYP303A1@ZIF-8, respectively;

[0036] G and H are the Zeta potential diagrams of ZIF-8 and dsNlCYP303A1@ZIF-8, respectively;

[0037] I is the XRD characterization diagram of ZIF-8 and dsNlCYP303A1@ZIF-8;

[0038] J is the element distribution map of dsNlCYP303A1@ZIF-8;

[0039] Figure 4 This is a functional analysis diagram of NlCYP303A1;

[0040] In the figure, A is the relative expression result of NlCYP303A1 48h after injection of dsGFP and dsNlCYP303A1;

[0041] B is the survival curve of brown planthopper after injection of dsNlCYP303A1 and dsGFP;

[0042] C is the lethal phenotype of brown planthopper after injection of dsNlCYP303A1 and dsGFP;

[0043] Figure 5 The figure shows the protective effect of ZIF-8 on dsNlCYP303A1 and its biological activity against brown planthopper;

[0044] In the figure, A is the result of feeding experiment to evaluate the effect of dsNlCYP303A1@ZIF-8 on NlCYP303A1 expression and survival rate of brown planthopper;

[0045] B-E are the results of NlCYP303A1 expression levels after 24, 48, 72 and 96 h of treatment in the feeding experiment;

[0046] F is the survival rate of brown planthoppers after 96 hours of treatment in the feeding experiment;

[0047] G is the result of evaluating the effect of dsNlCYP303A1@ZIF-8 on the survival of brown planthoppers by the rice seedling immersion method;

[0048] H is the result of the rice seedling immersion method to evaluate the relative expression of NlCYP303A1 after dsNlCYP303A1@ZIF-8 treatment for 48 h;

[0049] I is the result of evaluating the mortality of brown planthoppers after 96 hours of treatment with dsNlCYP303A1@ZIF-8 using the rice seedling immersion method. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0051] Example 1 Identification and sequence analysis of N1CYP303A1

[0052] 1. Full-length cloning and identification of NlCYP303A1

[0053] (1) Total RNA from brown planthoppers was extracted according to the instructions of RNAiso Plus total RNA extraction reagent from TaKaRa Company, and the first strand of cDNA was synthesized according to the procedures of HifairⅢ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigester plus) reverse transcription kit (YEASEN Company).

[0054] (2) A primer for amplifying the full length was designed at both ends of the coding region of the NlCYP303A1 gene. The sequences are shown in Table 1. The cDNA was used as a template and the NlCYP303A1 sequence fragment was cloned by RT-PCR. The PCR product was detected by agarose gel electrophoresis. The NlCYP303A1 gene fragment was recovered according to the operation method of the EZNA gel recovery kit. The recovered PCR product was detected by agarose gel electrophoresis, and a fragment of 1512 bp was recovered.

[0055] Table 1 Primer sequences for the full length of the N1CYP303A1 gene

[0056]

[0057] (3) Using TAKARA's Hieff Zero TOPO-TA simple Cloning Kit was used to connect the recovered fragments to the pESI-T vector. The ligation product was transformed into the Trelief TM5α competent cell strain, and then spread on a solid LB medium plate with ampicillin. After culturing for about 12 hours, a single white colony was picked and cultured in LB+Amp liquid medium. 2×Hieff PCR Mester Mix (YEASEN) was used for preliminary verification of bacterial liquid PCR, and the samples that were verified correctly were further sequenced to obtain the gene NlCYP303A1 with a fragment size of 1512 bp. The nucleotide sequence of the gene was sequenced and analyzed as shown in SEQ ID NO: 1.

[0058] 2. Sequence analysis of NlCYP303A1

[0059] The amino acid sequence of CYP303A1 from multiple species was analyzed using DNAMAN. Figure 1 As shown in Figures 1A and 1B, it was found to be highly conserved in multiple species.

[0060] The evolutionary tree was constructed using MEGA 11.0. Figure 1 As shown in C, the analysis found that NlCYP303A1 has a high degree of close relationship with LsCYP303A1 and SfCYP303A1.

[0061] Example 2: dsRNA-induced synthesis of N1CYP303A1 gene

[0062] (1) Based on the full-length sequence of NlCYP303A1, a fragment of about 500 bp was cloned using specific primers. The primer sequences are shown in Table 2. The nucleotide sequences of primers NlCYP303A1-F2 and NlCYP303A1R2 are shown in SEQ ID NOs: 2 and 3. The pESI-T-NlCYP303A1 plasmid was constructed using the Qingke ligation transformation kit. L4440 and pESI-T-NlCYP303A1 plasmids were digested with restriction endonucleases, and the target bands of the correct band size were recovered using the EZNA gel recovery kit.

[0063] Table 2 Primer sequences for dsRNA synthesis of N1CYP303A1 gene

[0064]

[0065] (2) The recovered fragments were ligated, and the ligation products were transformed into Trelief TM5α competent cell strains, and then spread on solid LB medium plates with ampicillin. After culturing for about 12 hours, single white colonies were picked and cultured in LB+Amp liquid medium. 2×Hieff PCR Mester Mix (YEASEN) was used for bacterial liquid PCR verification, and the plasmid was extracted using OMEGA's EZNAPlasmid Mini Kit to obtain the L4440-NlCYP303A1 plasmid vector.

[0066] (3) After the constructed L4440-NlCYP303A1 plasmid vector was transformed into HT115 competent cells, it was spread on a solid LB medium plate with ampicillin and cultured for about 12 hours. A single white colony was picked and cultured in LB+Amp liquid medium. A preliminary verification of the bacterial solution was performed using 2×Hieff PCR Mester Mix (YEASEN Company), and the bacterial solution with positive bacterial test was selected for sequencing and frozen.

[0067] (4) Use liquid LB medium to shake culture the HT115 strain containing the L4440-NlCYP303A1 plasmid at 37°C until OD 600 = 0.40, IPTG was added to a final concentration of 1 mM to induce expression, and the culture was continued at 37°C for 4 h. The total bacterial RNA was extracted, and the dsRNA (i.e., dsNlCYP303A1) obtained after purification was detected by spectrophotometer and 1% agarose gel electrophoresis for the concentration and quality of dsRNA ( Figure 2 ) and stored at -80℃ for later use.

[0068] Depend on Figure 2 It can be seen that there is a bright band at about 500 bp, and the size is consistent with the target band, indicating that dsNlCYP303A1 was successfully prepared.

[0069] Example 3 Indoor bioactivity assay of dsN1CYP303A1 against brown planthopper

[0070] dsGFP and dsNlCYP303A1 were injected into the 4th instar nymphs of brown planthoppers. The interference efficiency was detected by qRT-PCR. The results are shown in Figure 4 shown.

[0071] Depend on Figure 4 A shows that the relative expression level of NlCYP303A1 was significantly downregulated 48 hours after the fourth instar nymphs of brown planthopper were injected with dsNlCYP303A1 (P<0.05).

[0072] Depend on Figure 4B shows that at different time points after injection (24, 48, 72, 96 and 120 h), the survival rate of nymphs in the dsNlCYP303A1 treatment group was significantly lower than that in the dsGFP treatment group (P<0.05). 120 h after injection of dsNlCYP303A1, the survival rate of brown planthoppers decreased to 3.33%.

[0073] It is noteworthy that most of the 4th instar nymphs of brown planthoppers injected with dsNlCYP303A1 were unable to shed their old cuticles smoothly during molting and died with wrinkled new cuticles. To quantify the probability of this phenotype, the post-injection mortality of the nymphs was analyzed. Figure 4 C shows that 82.76% of the dead nymphs in the dsNlCYP303A1 injection group showed these abnormal death phenotypes. In contrast, no abnormal death was observed in the nymphs injected with dsGFP. The above results indicate that dsNlCYP303A1 treatment can significantly disrupt the molting process of brown planthoppers.

[0074] Example 4 Preparation of dsNlCYP303A1@ZIF-8 Nano-Nucleic Acid Pesticide System

[0075] This embodiment uses ZIF-8 to load dsNlCYP303A1, achieving target gene silencing while achieving the same level of control effect without adding pesticides, which is more environmentally friendly than the previous patent. The preparation method of the dsNlCYP303A1@ZIF-8 nucleic acid pesticide system loaded with dsNlCYP303A1 is as follows:

[0076] (1) Weigh 1 g Zn(NO3)2 6H2O, 13.86 g 2-methylimidazole and 80 mg dsNlCYP303A1;

[0077] (2) dissolving zinc nitrate hexahydrate and 2-methylimidazole in deionized water to obtain zinc nitrate hexahydrate solution (0.1 g / mL) and 2-methylimidazole solution (0.155 g / mL), respectively;

[0078] (3) dissolving dsNlCYP303A1 in zinc nitrate hexahydrate solution, mixing with 2-methylimidazole solution, and stirring at room temperature for 30 min to obtain a mixed solution;

[0079] (4) The mixed solution was centrifuged at 10,000 rpm for 10 min, the waste liquid was discarded, 40 mL of anhydrous ethanol solution was added, the mixture was vortexed on a vortexer for 10 min, and the mixture was centrifuged at 10,000 rpm for 10 min. The mixture was repeatedly washed with anhydrous ethanol for 3 times, the precipitate was collected, and dried in a vacuum to finally obtain the dsNlCYP303A1@ZIF-8 nanonucleic acid pesticide (hereinafter referred to as: dsNlCYP303A1@ZIF-8).

[0080] The dsNlCYP303A1@ZIF-8 nanopesticide was characterized by scanning electron microscopy, transmission electron microscopy, particle size distribution analysis, and transmission electron microscopy energy spectrum. Figure 3 shown.

[0081] Depend on Figure 3 As can be seen from A to 3D, TEM and SEM images indicate that the surface of dsNlCYP303A1@ZIF-8 is rough compared with ZIF-8.

[0082] Depend on Figure 3 E and Figure 3 It can be seen from F that DLS detection shows that ZIF-8 and dsNlCYP303A1@ZIF-8 particles are uniform, with particle sizes of 216.1nm and 385.0nm, respectively, and good dispersibility (PDI=0.118 and 0.164).

[0083] Depend on Figure 3 G and Figure 3 It can be seen from H that the negatively charged dsNlCYP303A1 was loaded into the positively charged ZIF-8, causing the zeta potential to change from 23.4 mV to -26.7 mV, proving that the loading of dsNlCYP303A1 was successful.

[0084] Depend on Figure 3 It can be seen from the XRD analysis of ZIF-8 and dsNlCYP303A1@ZIF-8 that the diffraction peak did not change after ZIF-8 was loaded with dsNlCYP303A1, which indicates that the loading of dsRNA has no significant effect on the crystal structure of the nanoparticles.

[0085] Depend on Figure 3 J It can be seen from the observation of dsNlCYP303A1@ZIF-8EDS spectrum that the phosphorus element of dsRNA exists in the delivery system.

[0086] The above results confirmed that the dsNlCYP303A1@ZIF-8 delivery system was successfully constructed.

[0087] Example 5 Determination of the biological activity of dsN1CYP303A1@ZIF-8 against brown planthopper

[0088] First, the effects of the cytotoxicity on the expression of NlCYP303A1 and the survival rate of brown planthoppers were evaluated through feeding experiments.

[0089] a. Blank treatment group (CK): brown planthoppers treated with 10% sucrose solution;

[0090] b. ZIF-8 treatment group: brown planthoppers treated with 10% sucrose solution containing ZIF-8 nanomaterials (the final concentration of ZIF-8 nanomaterials was 200 mg / L);

[0091] c. dsNlCYP303A1-treated group: brown planthoppers treated with 10% sucrose solution containing dsNlCYP303A1 (final concentration of dsNlCYP303A1 was 200 mg / L);

[0092] d. dsNlCYP303A1@ZIF-8 treated group: brown planthoppers treated with 10% sucrose solution containing dsNlCYP303A1@ZIF-8 (the final concentration of dsNlCYP303A1@ZIF-8 was 200 mg / L).

[0093] Depend on Figure 5 A~ Figure 5 It can be seen from F that there was no significant difference in the expression of NlCYP303A1 and the survival rate of brown planthoppers in the ZIF-8 treatment group and the control group, while in the dsNlCYP303A1@ZIF-8 treatment group, the expression level of NlCYP303A1 was significantly reduced at 24, 48, 72 and 96 h after treatment ( Figure 5 In contrast, in the treatment of feeding dsNlCYP303A1 alone, the expression of NlCYP303A1 was inhibited only 24 hours after treatment.

[0094] The above results indicate that encapsulating dsNlCYP303A1 in ZIF-8 can enhance its stability and persistence. After 96 h, it was found that dsNlCYP303A1@ZIF-8 treatment reduced the survival rate of brown planthoppers by 82.22%, while other treatments had no effect ( Figure 5 F).

[0095] The above experimental results show that dsNlCYP303A1@ZIF-8 has considerable potential in controlling brown planthoppers.

[0096] To further investigate whether dsNlCYP303A1@ZIF-8 can be used to directly control brown planthoppers on rice, its effect on the survival of brown planthoppers was evaluated by rice seedling dipping method.

[0097] a. Blank treatment group (CK): rice plants soaked in 0.1% Triton X-100 water for 30 seconds;

[0098] b. ZIF-8 treatment group: rice plants were immersed in 0.1% Triton X-100 water containing ZIF-8 nanomaterials (the final concentration of ZIF-8 nanomaterials was 500 mg / L) for 30 seconds;

[0099] c. dsNlCYP303A1 treatment group: rice plants were immersed in 0.1% Triton X-100 water containing dsNlCYP303A1 (final concentration of dsNlCYP303A1 was 500 mg / L) for 30 seconds;

[0100] d. dsNlCYP303A1@ZIF-8 treated group: rice plants were immersed in 0.1% Triton X-100 water containing dsNlCYP303A1@ZIF-8 (final concentration of dsNlCYP303A1@ZIF-8 was 500 mg / L) for 30 seconds.

[0101] Depend on Figure 5 G~ Figure 5 It can be seen that the relative expression level of NlCYP303A1 was significantly reduced after 48h of treatment with dsNlCYP303A1@ZIF-8 ( Figure 5 H), after 96h of treatment with dsNlCYP303A1@ZIF-8, the mortality rate of brown planthoppers reached 68.88% ( Figure 5 I).

[0102] The above results indicate that dsNlCYP303A1@ZIF-8 can be used as a nanonucleic acid pesticide for field control of brown planthoppers.

[0103] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A brown planthopper gene N1CYP303A1, characterized in that: The nucleotide sequence of the gene N1CYP303A1 is shown in SEQ ID NO:

1.

2. A primer pair for obtaining the brown planthopper gene N1CYP303A1 according to claim 1, characterized in that: The primer pair is NlCYP303A1-F2 and NlCYP303A1R2, and the nucleotide sequences thereof are shown in SEQ ID NOs: 2 and 3, respectively.

3. A recombinant vector L4440-N1CYP303A1, characterized in that: The recombinant vector is an expression vector containing the gene N1CYP303A1 according to claim 1, wherein the expression vector is L4440.

4. A host cell containing the recombinant vector L4440-N1CYP303A1 according to claim 3, characterized in that: The host cell is HT115 competent cell.

5. A method for inducing and extracting dsN1CYP303A1 using the host cell according to claim 4, characterized in that: The method is to culture host cells containing the recombinant vector L4440-N1CYP303A1 until OD 600 =0.40, add IPTG to a final concentration of 1 mM, induce expression, culture at 37°C with shaking for 4 h, extract total bacterial RNA, and purify the dsRNA obtained, namely dsNlCYP303A1.

6. Use of dsN1CYP303A1 prepared by the preparation method according to claim 5 in controlling brown planthopper.

7. A nano nucleic acid pesticide, characterized in that: In terms of weight, the raw materials of the nano-nucleic acid pesticide include 1 part of zinc nitrate hexahydrate, 13-15 parts of 2-methylimidazole and 0.06-0.1 parts of dsNlCYP303A1 prepared by the preparation method according to claim 5.

8. The nano nucleic acid pesticide according to claim 7, characterized in that: By weight, the raw materials of the nano nucleic acid pesticide include 1 part of zinc nitrate hexahydrate, 13.86 parts of 2-methylimidazole and 0.08 parts of dsNlCYP303A1.

9. A method for preparing a nano nucleic acid pesticide, characterized in that: The following steps are involved: (1) By weight, weigh 1 part of zinc nitrate hexahydrate, 13-15 parts of 2-methylimidazole, and 0.06-0.1 parts of dsNlCYP303A1 prepared by the preparation method of claim 5; (2) dissolving zinc nitrate hexahydrate and 2-methylimidazole in water to obtain zinc nitrate hexahydrate solution and 2-methylimidazole solution respectively; (3) dissolving dsNlCYP303A1 in a zinc nitrate hexahydrate solution, mixing with a 2-methylimidazole solution, and stirring at room temperature to obtain a mixed solution; (4) The mixed solution was centrifuged, the waste liquid was discarded, anhydrous ethanol was added to wash the precipitate three times, the precipitate was collected, and vacuum dried to obtain the nano-nucleic acid pesticide.

10. Use of the nano nucleic acid pesticide according to claim 7 in controlling brown planthopper.

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