Chitin synthase interacting protein CG8654 involved in chitin biosynthesis and its coding gene and application

Abstract

The application discloses a chitin synthase interacting protein CG8654 involved in chitin biosynthesis, a coding gene thereof and application. The application mines a chitin synthase interacting protein gene CG8654 in a model organism Drosophila melanogaster Drosophila melanogaster ) and finds that silencing the expression of the CG8654 gene in the epidermis of Drosophila melanogaster leads to larva death; knocking out the CG8654 in wings leads to abnormal chitin deposition, reduced chitin content and loss of layered structure; effectively silencing the homologous gene of the CG8654 in the last instar larvae of Ostrinia furnacalis and Tribolium castaneum leads to abnormal pupation and death. The results show that the CG8654 plays an important role in chitin synthesis, can provide a new target for pest control based on RNA interference, and has theoretical significance and broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a chitin synthase interaction protein involved in chitin biosynthesis, its encoding gene, and its applications. Background Technology

[0002] Harmful insects not only damage agricultural production but also spread various diseases. Pesticides are the main means of controlling pests, but their harm to human health and ecological safety, as well as the problem of pesticide resistance caused by long-term use, cannot be ignored. Continuous innovation of new pesticide targets and chemical entities is an important way to address these problems (Wu Jian, and Song Baoan. (2020) Current Status and Reflections on Green Pesticide Innovation and Target Research. China Science Foundation 34, 9). "Molecular Targets and Green Pesticide Molecular Design" was included in the Top 10 Frontiers of Engineering Research in the Agricultural Field in the report "Global Engineering Frontiers 2021" of the Chinese Academy of Engineering (Global Engineering Frontiers Project Group of the Chinese Academy of Engineering. (2021) Global Engineering Frontiers, Higher Education Press, Beijing).

[0003] Chitin, as a major structural component, constitutes the insect's epidermis, trachea, and midgut peritrophic membrane, among other structural tissues. These tissues play crucial roles throughout the insect's life cycle, preventing dehydration, defending against predators, protecting against pathogen invasion, and serving as supporting structures (Moussian, B. (2010) Recent advances in understanding mechanisms of insect cuticle differentiation. Insect Biochem Mol Biol40, 363-375). However, chitin is not found in higher plants and animals. Therefore, key proteins involved in chitin synthesis are potential targets for green pesticides. Although chitin synthase is the key enzyme in chitin synthesis, the complete process still requires the participation of various other proteins as cofactors, and some of these cofactors may have direct interactions with chitin synthase.Researchers have identified three interacting proteins of CHS1 (Kkv) in the fruit fly *Drosophila melanogaster*, including: choline transporter-like protein 2 (Ctl2) (Duan, Y., Zhu, W., Zhao, X., Merzendorfer, H., Chen, J., Zou, X., and Yang, Q. (2022) Choline transporter-like protein 2 interacts with chitin synthase 1 and is involved in insect cuticle development. *InsectBiochem Mol Biol 141, 103718*), and Sarco / Endoplasmic Reticulum Ca2+-ATPase (SERCA) (Zhu, W., Duan, Y., Chen, J., Merzendorfer, H., Zou, X., and Yang, Q. (2022) SERCA interacts with chitin synthase and participants in... Tissue-specific RNAi experiments have shown that these proteins play important roles in chitin synthesis, including cuticular chitin biogenesis in Drosophila (Insect Biochem Mol Biol 145, 103783) and fatty acid binding protein (Fabp) (Chen, J., Zou, X., Zhu, W., Duan, Y., Merzendorfer, H., Zhao, Z., and Yang, Q. (2022) Fatty acid binding protein is required for chitin biosynthesis in the wing of Drosophila melanogaster (Insect Biochem Mol Biol 149, 103845).

[0004] RNA interference (RNAi) technology is based on the degradation of homologous RNA mediated by double-stranded RNA (dsRNA), which can specifically reduce the expression of target genes. Compared with other gene-level manipulation methods, RNAi has a high efficiency in inhibiting gene expression and is relatively simple to operate, so it is widely used to study the function of certain genes in biological processes (Perrimon, N., Ni, JQ, and Perkins, L. (2010) Invivo RNAi: today and tomorrow. Cold Spring Harb. Perspect. Biol.2, a003640). In addition, RNAi also has great potential in pest control. Pest control through RNA interference has the following characteristics: high specificity to target organisms, easy degradation with no residue, and environmentally friendly and non-toxic. Summary of the Invention

[0005] In view of the importance of chitin biosynthesis in insects, this invention uses the model organism Drosophila melanogaster chitin synthase (Kkv) and three independent binding experiments: co-immunoprecipitation (co-IP), yeast two-hybrid (MYTH) and pull-down experiments to find that the protein CG8654 can directly interact with Kkv.

[0006] The binary expression Gal4 / UAS system, widely used in Drosophila melanogaster, is an important genetic research tool that enables tissue-specific RNAi targeting CG8654: silencing CG8654 gene expression in chitin-rich epidermis leads to pupal mortality in Drosophila (100% lethality). Non-lethal CG8654 knockout in the wings results in blistering in the wing midsection with significant changes in shape and size; further observation reveals abnormal chitin deposition, reduced chitin content, and loss of layered structure (100% phenotypic rate).

[0007] CG8654 possesses highly conserved homologous proteins in multiple insect orders, including various agricultural pests. Therefore, this invention further identified functionally conserved homologous proteins of CG8654 among agricultural pests such as the Asian corn borer (Lepidoptera) and the red flour beetle (Coleoptera) as OfOCT and TcOCT, respectively, using RNAi. Both of these homologous proteins share 44% amino acid sequence identity with CG8654, while their amino acid sequence similarities reach 63% and 62%, respectively.

[0008] Therefore, the present invention provides a chitin synthase interacting protein, characterized in that it has an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or has a similarity of more than 40%, preferably more than 60%, 70%, 80%, 90%, 95%, 98%, or 99%, and is a homologous protein with chitin synthase interaction ability derived from Drosophila melanogaster, Corn borer of Asia, or Red flour beetle.

[0009] Further, a gene encoding the chitin synthase interaction protein is provided. Preferably, it is a Drosophila melanogaster chitin synthase interaction protein gene having the nucleotide sequence shown in SEQ ID NO: 2; or an Asian corn borer chitin synthase interaction protein gene having the nucleotide sequence shown in SEQ ID NO: 4; or a Red flour beetle chitin synthase interaction protein gene having the nucleotide sequence shown in SEQ ID NO: 6.

[0010] The present invention also provides expression vectors and recombinant cells containing the aforementioned genes.

[0011] Furthermore, the present invention provides dsRNA targeting the aforementioned gene. More specifically, the dsRNA has a nucleotide sequence as shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0012] This invention also provides the use of the aforementioned gene as a target in pest control. Specifically, the pest is a lepidopteran such as the Asian corn borer, or a coleopteran such as the red flour beetle.

[0013] This invention also provides the use of the dsRNA in pest control. Specifically, the pests are Lepidoptera such as the Asian corn borer, or Coleoptera such as the red flour beetle.

[0014] More specifically, the application of synthesized OfOCT dsRNA in the control of the Asian corn borer was investigated. The synthesized dsRNA was injected into the body cavity of 5th instar larvae of the Asian corn borer using a microsyringe. Results showed that, compared to the control, OfOCT dsRNA caused abnormal pupation and abdominal malformation in larvae (phenotypic rate 12.5%), ultimately leading to death. The application of synthesized TcOCT dsRNA in the control of the red flour beetle was also investigated: the synthesized dsRNA was injected into the body cavity of 20-day-old red flour beetle larvae using a microinjection device. Results showed that, compared to the control, TcOCT dsRNA caused abnormal pupation and wing malformation in larvae (phenotypic rate 33.9%), ultimately leading to death.

[0015] Therefore, selecting the chitin synthase interaction protein gene as the target and utilizing its important biological functions for pest control is safe, efficient, environmentally friendly, non-toxic and harmless to humans, and a novel specific molecular target with broad application prospects in the field of pest control. Attached Figure Description

[0016] Figure 1 Schematic diagram of the distribution of Kkv antigen recognition sites.

[0017] Figure 2 : Schematic diagram of immunoprecipitation procedure.

[0018] Figure 3A : Schematic diagram of the truncated Kkv body.

[0019] Figure 3B Results of yeast two-hybridization.

[0020] Figure 4 Pull-down experiment. Black triangles indicate protein locations.

[0021] Figure 5A The effect of RNAi on the relative expression level of CG8654 in the epidermis.

[0022] Figure 5B Effect of wing RNAi on the relative expression level of CG8654.

[0023] Figure 6 In different organizations CG8654 The effect of gene silencing on Drosophila development. A: Epidermis; B: Wings. Scale bar: 500µm. n represents the total number of statistics, and the percentage represents the proportion of the phenotype.

[0024] Figure 7 : ap-Gal4 driver CG8654 Effects of gene silencing on Drosophila wing development. A: Eosin staining showing the effect of CG8654 interference on wing permeability, scale bar: 200 µm; B: Cross section of Drosophila wing, chitin stained with calcium fluorescent white. The first longitudinal vein is shown in the black box in the left image and magnified in the right image. Images were converted into color-coded LUTs, where different levels of fluorescence signal are represented by different colors, black scale bar: 100 µm, white scale bar: 5 µm; B: Transmission electron microscopy image of the Drosophila wing vein epidermis, scale bar: 500 nm.

[0025] Figure 8A Effect of RNAi on the relative expression level of OfOCT (dsOfOCT synthesized by injection in 5-year-old Asian corn borers).

[0026] Figure 8BPhenotypic diagrams of the control and experimental groups in the dsOfOCT injection experiment on 5-year-old Asian corn borers (scale bar: 3 mm).

[0027] Figure 9A Effect of RNAi on relative expression of TcOCT at 20 days old (dsTcOCT synthesized by injection of *Strombus haematocephala*).

[0028] Figure 9B Phenotypic diagrams of the control and experimental groups in the experiment of dsTcOCT synthesized by injection of Trichoderma rubescens (scale bar: 500 µm). Detailed Implementation

[0029] The present invention will be further described below through specific embodiments, but these embodiments do not constitute a limitation thereof.

[0030] Example 1: Immunoprecipitation 1. Fruit fly breeding The Drosophila melanogaster strain W1118 was reared in a constant temperature and humidity incubator under the following conditions: temperature 25 ℃, relative humidity 60%, and light-to-dark ratio 12:12. The artificial feed formula consisted of 100 g corn flour, 130 g sucrose, 10 g agar powder, and 20 g Angel yeast powder added to 1.2 L distilled water; 0.75 g benzoic acid and 1.25 ml propionic acid were used as preservatives. Pupae were collected 48 hours after pupation, flash-frozen in liquid nitrogen, and stored at -80 ℃ for later use.

[0031] 2. Preparation of specific antibodies 1) Expression and purification of antigens Based on the experimental requirements of immunoprecipitation, a truncated Kkv variant with a recombinant expression size of approximately 15 kDa was selected as the antigen. Based on conservation, the final selected regions were the catalytic domain and the coiled-coil region. Specific distribution details are as follows. Figure 1As shown. The fusion proteins Kkv-GT (amino acids: 661-823) and Kkv-CC (amino acids: 1144-1273) with a 6×His tag appended to the C-terminus were expressed in *E. coli* BL21 (DE3) using the vector pET-28a (+). Specifically, to obtain the expression vectors, the cDNA fragments encoding Kkv-GT and Kkv-CC were synthesized by Sino-American Taihe Pharmaceutical Co., Ltd. (Beijing, China) after codon optimization. The expression strain was first activated and expanded in 5 ml of LB medium containing kanamycin (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) at 37 °C with shaking at 200 rpm / min overnight. The next day, the bacterial culture was added to 0.5 L of LB medium containing kanamycin at a ratio of 1:100 and cultured at 37 °C until OD600 ≥ 0.6. Expression was induced overnight at 16 °C after adding 0.4 mM isopropyl galactothioglycoside (IPTG) as an inducer. Kkv-GT and Kkv-CC were finally purified using a HisTrap HP (GE, USA) affinity chromatography column.

[0032] 2) Preparation of polyclonal antibodies Antibody preparation and purification were performed by Shanghai Jier Biochemical. The specific procedure was as follows: 1 ml of PBS (Phosphate Buffered Saline; 8 mM Na2HPO4, 136 mM NaCl, 2 mM KH2PO4, 2.6 mM KCl; pH 7.4) containing 400 μg of purified antigen (Kkv-GT or Kkv-CC) was mixed with 1 ml of Freund's complete adjuvant and subcutaneously injected into the back of rabbits every two weeks. After the fourth immunization, the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). The rabbit antiserum was purified by 50% saturated ammonium sulfate precipitation and DEAE-Sepharose column chromatography to obtain the rabbit IgG fraction. The concentration of the purified antibody was determined using a UV spectrophotometer, and the antibody was stored in PBS containing 0.02% sodium azide.

[0033] 2. Immunoprecipitation and mass spectrometry analysis Two g of Drosophila pupae were placed in a mortar and ground completely with liquid nitrogen. The resulting fragments were then dissolved in TBS buffer (50 mM Tris, 150 mM NaCl; pH 7.5) containing a protease inhibitor mixture (Thermo Fisher Scientific, USA). The lysate was centrifuged at low speed (700×g, 4 ℃, 10 min) to remove undisturbed cells, nuclei, and other debris. The collected supernatant was then further centrifuged at high speed (200,000×g, 4 ℃, 60 min). The precipitate was collected and washed with TBS buffer, then resuspended in solubilization buffer (TBS buffer + 0.5% n-dodecyl-D-maltose (DDM)). After incubation on ice for 30 minutes, the dissolved membrane fraction was collected by high speed centrifugation (200,000×g, 4 ℃, 60 min). An appropriate amount of the dissolved membrane fraction was used for Western blotting identification using anti-Kkv-GT and anti-Kkv-CC antibodies. Next, the Kkv antibody was coupled to Amino-LinkPlus cross-linking resin (Thermo Fisher Scientific) to prepare affinity purification resin; rabbit-derived IgG was also coupled to Amino-LinkPlus cross-linking resin as a control group. The dissolved membrane components and affinity purification resin were incubated at 4°C with gentle stirring for 2 h. After thorough washing with washing buffer (TBS buffer + 0.1% DDM), the affinity column was eluted with elution buffer (100 mM Glycine-HCl, pH 2.8). The experiment was performed in triplicate, with the procedure as follows: Figure 2 As shown.

[0034] Mass spectrometry analysis was performed by OE Bio (Shanghai, China). For each sample, 10 μg of total protein was dried and resuspended in 100 mM Tris-HCl (pH 8.5) supplemented with 8 M urea. Samples underwent further reduction, acetamination, and trypsin digestion. Samples were analyzed using a QExactive system (Thermo Fisher Scientific) equipped with an Easy-nLC 1000 HPLC system. Raw files were processed on Proteome Discoverer v.1.2 (Thermo Fisher Scientific) using Mascot v.2.2 (Matrix Science, USA) as the search engine. Kkv interacting proteins were required to meet the following criteria for data analysis: 1) The number of specific peptides identified in the eluents of both experimental groups in each biological replicate was greater than or equal to 2, and at least three times the number of specific peptides identified in the control group eluent; 2) Criterion 1 was met in at least two biological replicates. This method was used to initially screen and identify Kkv, demonstrating the reliability of the antibody and experimental method. Based on the mass spectrometry analysis results, a protein that may interact with Kkv was identified. Its FlyBase database number is CG8654. Due to the lack of functional studies on it for a long time, it has not yet been formally named.

[0035] Example 2: Yeast Two-Hybrid Experiment 1. cDNA template preparation Total RNA was extracted from Drosophila melanogaster pupae using the MiniBEST Universal RNA Extraction Kit (Takara Bio, Japan). Reverse transcription of mRNA was performed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit for RT-PCR (Takara Bio), with 5 μg of total RNA as a template and 1 μl of Oligo dT Primer used. The resulting cDNA was diluted 1:10 and frozen for later use. The cDNA clone of CG8654 from Drosophila melanogaster was purchased from the Drosophila Genomics Resource Center (DGRC), clone number GH28654.

[0036] 2. Splitting ubiquitin membrane yeast two-hybrid (MYTH) The Split-ubiquitin membrane yeast two-hybrid (MYTH) system is based on the original nucleoprotein yeast two-hybrid system, introducing a pair of artificially separated ubiquitin truncated bodies to mediate the recognition of protein-protein interaction signals. The MYTH experiment was performed using the DUALmembrane starter kit (Dualsystem, Switzerland). Compared to the conventional soluble protein yeast two-hybrid system, the MYTH system is relatively complex, requiring at least one of the N-terminus or C-terminus of the bait protein and prey protein to be located in the cytoplasm, because the fused ubiquitin can only be recognized and trigger subsequent reactions when it is located in the cytoplasm. To meet this requirement, using specified primers (Table 1), the truncated sequences of three kkvs—kkv-NTR (1-1617), kkv-CD (1597-2733), and kkv-CTR (2713-4845)—were amplified by PCR. These sequences were then inserted into the bait vectors pBT3-STE, pDHB1, and pBT3-N, respectively, using EasyGeno Assembly Mix. Figure 3A ). use Figure 3A The full-length cDNA sequence of CG8654 was amplified by PCR using the listed primers and cloned into the pPR3-N vector using EasyGeno Assembly Mix. The bait and prey plasmids were then co-transformed into NMY51 competent cells. Positive transformants were screened on SD-Trp-Leu medium (DDO) (Takara Bio), and protein-protein interactions were detected on SD-Trp-Leu-His medium (TDO) (Takara Bio) and / or SD-Trp-Leu-His-Ade medium (QDO) (Takara Bio). A positive control was a combination of the bait plasmid and pOst1-NubI. A negative control was a combination of the bait plasmid and pPR3-N. Yeast was grown at 29 °C for 4 days, and growth was recorded by photograph. The final MYTH experiment results demonstrated the interaction between Kkv-NTR and CG8654. Figure 3B ).

[0037] Table 1 Primers used in this embodiment Example 3: Pull-down Experiment The cDNA sequences encoding kkv and CG8654 were codon-optimized, synthesized by GenScript (Nanjing, China), and cloned into the pcDNA3.1 plasmid to construct expression vectors kkv-Strep and CG8654-Flag, respectively. In the experimental group, Kkv-Strep and CG8654-Flag were co-expressed in HEK293F cells (September Pharma, China) using SMM 293-TII expression medium (September Pharma). In the control group, Kkv-Strep and CG8654-Flag were expressed separately in HEK293F cells (September Pharma). After culturing at 37 °C for 48 h, cells were collected by centrifugation. Cells were resuspended in TBS buffer, and after adding protease inhibitors, the cells were sonicated and then collected by ultracentrifugation (200,000 × g, 4 °C, 60 min). The precipitate was dissolved in TBS containing 0.5% DDM, incubated at 4°C for 2 h, and then centrifuged at high speed (18000 ×g, 4°C, 30 min) to collect the supernatant. A portion of the supernatant was kept as a control, while the remainder was incubated with Anti-Flag G1 affinity resin (GenScript) and Strep-Tactin®XT affinity resin (IBA, Germany), respectively. After the affinity resins were thoroughly washed with TBS buffer containing 0.1% DDM, SDS loading buffer was added and the mixture was boiled. The samples were then directly subjected to SDS-PAGE electrophoresis. Monoclonal antibodies targeting the Strep and Flag tags (GenScript) were used in the Western blotting of proteins.

[0038] In the "Flag pull-down" experimental group, CG8654-Flag was used as the decoy, and detection was performed using anti-Flag and anti-Strep antibodies. Conversely, in the "Strep pull-down" experimental group, Kkv-Strep was used as the decoy, and detection was performed using anti-Flag and anti-Strep antibodies. Both pull-down experiments confirmed that CG8654 is an interacting protein with Kkv. Figure 4 ).

[0039] Example 4: Tissue-specific RNAi in Drosophila melanogaster 1. Sources and rearing of fruit fly strains The Gal4-driven subspecies 69B-Gal4 (widely expressed in the epidermis, BDSC1774) and ap-Gal4 (expressed on the dorsal side of the wing, BDSC3041) were purchased from the Bloomington Drosophila Stock Centre (BDSC). The transgenic UAS-RNAi strain UAS-CG8654-RNAi (hpRNA encoding CG8654, VDRC100112) was purchased from the Vienna Drosophila Resource Centre (VDRC). All Drosophila melanogaster strains were reared in a temperature and humidity controlled incubator under the following conditions: temperature 25 °C, relative humidity 60%, and light ratio 12:12.

[0040] 2. Fruit fly hybridization and temperature control Male flies from the UAS-CG8654-RNAi strain were crossed with virgin flies from the 69B-Gal4 and ap-Gal4 driven strains, respectively. All parents and offspring were cultured at 25 °C. The F1 genotypes were 69B > CG8654-RNAi and ap > CG8654-RNAi, respectively. Wild-type (wt) male flies and virgin flies from the 69B-Gal4 / ap-Gal4 strains served as a control group, with the same treatment conditions as the experimental groups.

[0041] 3. Sample preparation and RT-qPCR The epidermis of Drosophila larvae in the experimental group 69B > CG8654-RNAi and the corresponding control group were dissected. Twenty larvae were placed in each centrifuge tube, with three tubes used as biological replicates. After flash freezing in liquid nitrogen, the samples were stored at -80°C. Additionally, the wing buds of Drosophila pupae in the experimental group ap > CG8654-RNAi and the corresponding control group were dissected. Twenty pupae were placed in each centrifuge tube, with three tubes used as biological replicates. After flash freezing in liquid nitrogen, the samples were stored at -80°C.

[0042] Total RNA was extracted from the collected experimental and control samples using the MiniBEST Universal RNA Extraction Kit (Takara Bio). The EasyScript® All-in-One First-Strand cDNA SynthesisSuperMix for qPCR (TransGold) was used to synthesize the first-strand cDNA. The resulting cDNA was diluted 1:5 and used for RT-qPCR analysis. Gene expression levels in the experimental and control groups were analyzed by qPCR using the PerfectStart® Green qPCR SuperMix (TransGold) on an Applied Biosystems 7500 real-time quantitative PCR system (Thermo Fisher Scientific). The relative expression level of the CG8654 gene was calculated using the 2–∆∆Ct method, with the expression level of the internal reference gene ribosomal protein 49 (Rp49) as the standard. The primers used in this experiment are as follows: All experiments used three biological replicates, each containing three technical replicates. RT-qPCR results showed that, compared with the control group, the relative expression level of CG8654 was significantly reduced in the experimental group 24 h after pupation. Figure 5A and Figure 5B ).

[0043] 4. Observation and imaging of fruit fly phenotypes 1) Phenotypic observation Fruit fly pupae or adults three days after emergence were collected and anesthetized with ice. Imaging was performed using a stereomicroscope (SZX16, Olympus, Japan). After silencing CG8654 in the epidermis, the experimental group of fruit flies could hatch normally as larvae and pupate; however, they died successively during the pupal stage and failed to metamorphose into adults. Figure 6 (A). Driven by ap-Gal4, CG8654 expression was interfered on the dorsal side of the wing (ap > CG8654-RNAi). All fruit flies in the experimental group showed similar phenotypes in their wings; compared with normal wings, their wings had bulges in the middle and showed significant changes in shape and size. Figure 6 (B)

[0044] 2) Eosin staining of wings Fruit flies that had emerged three days prior were collected and anesthetized with CO2. The whole fruit flies were immersed in 0.5% eosin dye (Eosin Y; Solarebro, China) containing 0.1% Triton X-100 and incubated at 55 °C for 30 min. After staining, the fruit flies were washed three times with distilled water, and their wings were removed with tweezers. Immediately afterward, images were created using a stereomicroscope (SZX16).

[0045] In the control group, only the second and third anterior chambers of the wings in the posterior half were infiltrated with eosin dye. However, in the experimental group (ap > CG8654-RNAi), all areas of the wings of all fruit flies were infiltrated with eosin dye. Figure 7 (A) Increased wing permeability indicates that inhibiting CG8654 expression affects the barrier function of the wing epidermis.

[0046] 3) Frozen sections and calcium fluorescent staining Fruit flies that had emerged three days prior were collected and anesthetized with CO2. The flies were dissected on a plate continuously releasing CO2. Wings were removed with forceps and fixed overnight at 4°C in a PBS buffer containing 4% (w / v) paraformaldehyde and 0.1% Triton X-100, pH 7.4. After fixation, the flies were washed three times with PBST buffer (PBS buffer containing 0.1% Triton X-100), 20 min each time.

[0047] Transfer the cleaned wings to a 30% sucrose solution (30 g sucrose dissolved in PBS buffer, brought to a final volume of 100 ml) and incubate at room temperature (25-27 °C) for 1 h. Place the wings into a cubic mold containing a 55 °C solution of 10% gelatin (Gel Strength 300 Type A, Sigma-Aldrich, USA) and freeze at 4 °C to accelerate solidification. Add cryo-section embedding medium (Optimal Cutting Temperature Compound, OCT, Sakura, Japan) to submerge the sample block, and then quickly freeze the mold at -80 °C.

[0048] The frozen sample was removed from the mold and fixed onto the sample holder of a cryostat (NX50, Thermo Fisher Scientific) using cryosection embedding medium at -25 °C, with a section thickness of 10 µm. After drying at room temperature for 24 hours, the sections were washed three times with PBST buffer. Calcium fluorescent white (CFW, 1 mg / ml, Sigma-Aldrich) was then added to the sample and stained for 5 min. After washing three times with PBST buffer, 20 µl of 50% glycerol was added to the sample, covered with a coverslip, and sealed with nail polish. Fluorescence imaging of the CFW in the sample was performed using a 405 nm laser in a laser confocal microscope (LSM 880, Zeiss, Germany).

[0049] In the experimental group of fruit flies with CG8654 interference (ap > CG8654-RNAi), the chitin content on the dorsal side of the first longitudinal vein of the wing was also reduced to varying degrees. Figure 7 (Middle B, dorsal side).

[0050] 4) Ultrathin section and transmission electron microscopy observation Fruit flies that had emerged three days prior were collected and anesthetized with CO2. The flies were dissected on a CO2-containing plate, and wings were removed with forceps and placed in 2.8% glutaraldehyde (Soleb) containing 0.1% Triton X-100 for fixation at 4 °C for 48 h. The collected wings were washed three times with PBST and then fixed with 1% osmium tetroxide at 4 °C for 3 h. Subsequently, the fixed wings were washed three more times with PBS, followed by stepwise dehydration in a gradient of acetone (50%, 70%, 80%, 90%, 100%). The dehydrated wings were then embedded in Epon 812 embedding medium at room temperature for 2 h. Semi-thin sections were cut, and after screening and localization, ultrathin sections were prepared from designated areas. Ultrathin sections were collected using a copper mesh and stained with 4% uranium acetate. The ultrathin sections were observed and imaged using a transmission electron microscope (JEM-1200EX, JEOL, Japan).

[0051] In the control group, the epidermis at the longitudinal vein location of the wings consisted of the protoepidermis and the epidermis, with a distinct chitinous lamellar structure present in the protoepidermis. In the wings of fruit flies interfered with by CG8654 (ap > CG8654-RNAi), the chitinous lamellar structure of the protoepidermis was lost. Figure 7 (C, dorsal side) Example 5: Lethal Experiment of OfOCT Gene dsRNA in Asian Corn Borer 1. Synthesis of OfOCT gene-specific dsRNA in Asian corn borer First, based on the amino acid sequence of Drosophila melanogaster CG8654 (SEQ ID NO: 1), a BLAST program was run in the NCBI database to find the amino acid sequence of the homologous protein from the Asian corn borer (SEQ ID NO: 3), which has the highest similarity to Drosophila melanogaster. The amino acid sequence similarity between the two is 44%, and the sequence similarity is as high as 63%. The corresponding base sequence was also identified (SEQ ID NO: 4).

[0052] Next, the required dsRNA sequence (SEQ ID NO: 8) was designed, and the sense strand of the dsRNA, plus approximately 100 bp of subsequent sequence and the antisense strand, were synthesized and ligated into the pET-28a(+) plasmid to form a neck-loop hairpin structure. The ligation restriction sites were XbaI–XhoI. After plasmid transformation, single colonies were picked and cultured until OD600 = 0.6. IPTG was added to induce dsRNA expression. After induction, RNA was extracted using ethanol fixation. The size of the target band was detected by agarose gel electrophoresis, and the product concentration was detected using Nanodrop2000.

[0053] 2. Injection of the OfOCT gene dsRNA into the Asian corn borer Thirty healthy, uniformly sized fifth-instar larvae on day 2 were selected for the experiment. 2 μl (10 μg) of dsRNA (SEQ ID NO: 8) was injected into the space between the second and third abdominal segments on the lateral abdomen of the larvae using a 10 μl microsyringe. Thirty larvae were selected as a control group. The same volume and concentration of dsGFP were injected into the control group. The injected larvae were then placed in a 26℃ constant-temperature biochemical incubator (light-to-illuminance ratio 16:8, temperature 26±1℃, humidity 70%) and fed fresh artificial feed daily.

[0054] 3. Silent Efficiency Detection Six larvae from each of the experimental and control groups were collected after injection. Total RNA was extracted from two larvae per group and reverse transcribed into first-strand cDNA. Three biological replicates were set up for each group, with three larvae per replicate. The relative expression levels of the target gene and the housekeeping gene ribosomal protein S3 (RpS3) were detected using Real-time PCR, and the silencing efficiency was calculated. The primers used in this experiment are as follows: All experiments used three biological replicates, each containing three technical replicates. Compared with the control group, the expression level of the target gene was significantly reduced in the experimental group after injection of dsRNA. Figure 8A ).

[0055] 4. Phenotypic observation after dsRNA injection In the control group, 5th instar larvae injected with dsGFP could pupate normally and molt into adults. In contrast, OfOCT's dsRNA caused abnormal pupation and abdominal malformation in larvae (phenotype rate 12.5%), ultimately leading to death. Figure 8B ).

[0056] Example 6: Lethality experiment of TcOCT gene dsRNA of Red Flour Beetle 1. Synthesis of TcOCT gene-specific dsRNA in the red flour beetle First, based on the amino acid sequence of Drosophila melanogaster CG8654 (SEQ ID NO: 1), a BLAST program was run in the NCBI database to find the amino acid sequence of the homologous protein in Beetle erythroptera (SEQ ID NO: 5), which has the highest similarity to Drosophila melanogaster. The amino acid sequence similarity between the two is 44%, and the sequence similarity is as high as 62%. The corresponding base sequence was also identified (SEQ ID NO: 6).

[0057] Next, the required dsRNA sequence (SEQ ID NO: 9) was designed, and the sense strand of the dsRNA, plus approximately 100 bp of subsequent sequence and the antisense strand, were synthesized and ligated into the pET-28a(+) plasmid to form a neck-loop hairpin structure. The ligation restriction sites were XbaI–XhoI. After plasmid transformation, single colonies were picked and cultured until OD600 = 0.6. IPTG was added to induce dsRNA expression. After induction, RNA was extracted using ethanol fixation. The size of the target band was detected by agarose gel electrophoresis, and the product concentration was detected using Nanodrop2000.

[0058] 2. Injection of TcOCT gene dsRNA from *Erythropus erythropus* Thirty healthy, uniformly sized, 20-day-old final-instar larvae of the red flour beetle were selected. 200 nL (1 μg) of synthesized dsRNA (SEQ ID NO: 9) was injected via microinjection into the space between the second and third abdominal segments of the larvae. Thirty larvae were simultaneously selected as a control group. The same volume and concentration of dsGFP were injected into the control group. The injected larvae were then placed in a 30℃ constant-temperature biochemical incubator (light ratio 12:12, temperature 30±1℃, humidity 70%) and fed whole wheat flour (containing 5% dry yeast powder) daily.

[0059] 3. Silent Efficiency Detection Nine larvae from each of the experimental and control groups were collected after injection. Total RNA was extracted from groups of three larvae each and reverse transcribed into first-strand cDNA. Three biological replicates were set up for each group, with three larvae per replicate. Real-time PCR was used to detect the relative expression levels of the target gene and the housekeeping gene RpS3, and the silencing efficiency was calculated. The primers used in this experiment are as follows: All experiments used three biological replicates, each containing three technical replicates. Compared with the control group, the expression level of the target gene was significantly reduced in the experimental group after injection of dsRNA. Figure 9A ).

[0060] 4. Phenotypic observation after dsRNA injection In the control group, 20-day-old larvae injected with dsGFP could pupate normally and molt into adults. In contrast, TcOCT dsRNA caused abnormal pupation and wing malformation in larvae (phenotype rate of 33.9%), ultimately leading to death. Figure 9B ).

Claims

1. A dsRNA, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:8 or SEQ ID NO:

9.

2. The use of a gene for a chitin synthase interaction protein as a target in pest control, wherein the pest is the Asian corn borer; The gene encoding the chitin synthase interaction protein is shown in the amino acid sequence of SEQ ID NO:

3.

3. The use of a gene for a chitin synthase interaction protein as a target in pest control, wherein the pest is the red flour beetle; The gene encoding the chitin synthase interaction protein is shown in the amino acid sequence of SEQ ID NO:

5.

4. The use of dsRNA in pest control, wherein the pest is the Asian corn borer; characterized in that, The nucleotide sequence of the dsRNA is shown in SEQ ID NO:

8.

5. The use of a dsRNA in pest control, wherein the pest is the red flour beetle; characterized in that, The nucleotide sequence of the dsRNA is shown in SEQ ID NO: 9.

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