CYP9E22 gene and application thereof in regulating and controlling cyhalothrin resistance of periplaneta americana
By regulating the CYP9E2_2 gene of the American cockroach, the problem of resistance to cyhalothrin in the American cockroach was solved, achieving effective control of the insecticide. This provides a technical means of gene regulation and protein inhibition, which can be applied in the field of pest management.
Patent Information
- Application Number
- CN202511091522.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-05
- Publication Date
- 2025-11-11
AI Technical Summary
American cockroaches have developed resistance to lambda-cyhalothrin, and existing technologies are insufficient to effectively manage this resistance, thus affecting pest control outcomes.
By knocking out or inhibiting the CYP9E2_2 gene, the detoxification ability of American cockroaches against cyhalothrin was reduced. CRISPR/Cas9 technology, dsRNA, siRNA and other methods were used to regulate gene expression, CYP9E2_2 protein inhibitors were developed, and CYP9E2_2 protein was expressed in host cells using recombinant vectors to study its detoxification mechanism.
It effectively reduces the resistance of American cockroaches to cyhalothrin, restores the insecticide's killing effect, and provides a resistance management strategy for the preparation of CYP9E2_2 protein and screening of inhibitors.
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Figure CN120924557A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, and particularly relates to... CYP9E2_2 Genes and their application in regulating American cockroach resistance to cyhalothrin. Background Technology
[0002] American cockroaches are common pests that can transmit many diseases to humans in daily life by carrying pathogens, bacteria, and parasite eggs, such as Enterobacteriaceae. Enterobacterium ), Pseudomonas spp. ( Pseudomonas Cockroaches, including those carrying bacteria such as vesicles, can adversely affect multiple systems in humans, including the urinary, digestive, and respiratory systems. When humans accidentally come into contact with the secretions and excrement of cockroaches, the bacteria on their skin can cause allergic reactions, such as skin rashes, allergic rhinitis, and itching. More seriously, it can even trigger asthma.
[12] In addition, it can cause gastrointestinal diseases, especially when people accidentally ingest cockroach excrement, leading to food poisoning and diarrhea. In extreme cases, it can become a foreign object in the external auditory canal or even the bronchus, endangering human health. Furthermore, the American cockroach is also an "industrial pest," appearing in various industries and causing damage to transportation, textiles, food, pharmaceuticals, electronics, tourism, and other sectors, with the damage becoming increasingly severe.
[0003] Chemical insecticides remain the most common and effective pest management method for controlling American cockroaches. Common insecticides include nereistoxins, pyrethroids, diamides, and neonicotinoids, and their effects are relatively significant. Lambda-cyhalothrin (FCC) has good control effects on American cockroaches and has become the main means of controlling them in many areas. However, with the continued use of CFC, some American cockroaches have gradually developed resistance to it. Proactively implementing resistance management strategies before widespread resistance to CFC develops in American cockroaches is of great significance for the sustainable use of CFC. Summary of the Invention
[0004] The present invention aims to provide a method for regulating the resistance of American cockroaches to cyhalothrin. CYP9E2_2 Gene.
[0005] In this plan CYP9E2_2 Gene, CYP9E2_2 The gene sequence is shown in SEQ ID NO.1.
[0006] SEQ ID NO.1:
[0007] Research has found that, CYP9E2_2 The gene is involved in the detoxification process of the American cockroach against the highly effective cyhalothrin; therefore, the present invention also claims protection for the following: CYP9E2_2 Application of genes in regulating resistance of American cockroaches to cyhalothrin.
[0008] Knockout CYP9E2_2 Gene knockout (using technologies such as CRISPR / Cas9) can reduce the resistance of American cockroaches to cyhalothrin. By knocking out this gene (reducing its function), the detoxification ability of American cockroaches to cyhalothrin can be directly weakened, making them sensitive to the insecticide again.
[0009] The encoding possesses the above CYP9E2_2 The protein of a gene or its active fragment. CYP9E2_2 Proteins and their active fragments, as functional entities, can be used to determine their detoxification efficiency against cyhalothrin (such as metabolic rate, substrate specificity, etc.) through in vitro enzyme activity assays, revealing the molecular mechanism of resistance. At the same time, as drug screening targets, they can be used for high-throughput screening of compounds that can inhibit its activity (such as small molecule inhibitors).
[0010] contain CYP9E2_2 Recombinant vectors or host cells are used to reconstitute genes. Recombinant vectors (such as plasmids and viral vectors) enable stable expression of the gene in a heterologous host (such as E. coli or insect cells), while the host cell serves as a "biofactory" for large-scale in vitro production. CYP9E2_2 Proteins that meet experimental needs such as enzyme activity detection, structural analysis, and inhibitor screening.
[0011] Targeted silencing CYP9E2_2 dsRNA, siRNA, or expression vectors. dsRNA and siRNA can be specifically inhibited using RNA interference technology. CYP9E2_2 Gene transcription or translation reduces gene expression levels; expression vectors can continuously generate silencing molecules in vivo, prolonging the silencing effect.
[0012] CYP9E2_2 Inhibitors. Inhibitors can directly bind to... CYP9E2_2 The protein inhibits its detoxification activity (such as preventing it from metabolizing cyhalothrin), thus preventing resistant American cockroaches from degrading the insecticide and restoring the insecticide's killing effect.
[0013] Expression or knockout CYP9E2_2 Transgenic insect cells, model organisms, or microorganisms expressing this gene can be used to simulate a high-resistance state and study its detoxification mechanism; models with the gene knocked out can simulate a low-resistance state and allow for comparative analysis of resistance-related phenotypic changes. Attached Figure Description
[0014] Figure 1 American cockroach CYP9E2_2 Expression levels at different parts of the gene.
[0015] Figure 2 American cockroach CYP9E2_2 Genetic phylogenetic tree.
[0016] Figure 3 for CYP9E2_2 Protein signal peptide prediction results.
[0017] Figure 4 American cockroach CYP9E2_2 Graph showing the analysis of protein hydrophilicity coefficients.
[0018] Figure 5 American cockroach CYP9E2_2 Protein secondary structure prediction diagram; in the diagram, blue represents α-helices, purple represents random coils, red represents extended chains, and green represents β-sheets.
[0019] Figure 6 American cockroach CYP9E2_2 Predicted tertiary structure diagram of protein.
[0020] Figure 7 American cockroach CYP9E2_2 Predicted map of conserved protein domains.
[0021] Figure 8 American cockroach CYP9E2_2 Predicted transmembrane domains of proteins.
[0022] Figure 9 American cockroach CYP9E2_2 Protein phosphorylation prediction results; phosphorylation sites, S for serine, T for threonine, Y for tyrosine.
[0023] Figure 10 American cockroach CYP9E2_2 Figure showing the predicted results of protein glycosylation. Detailed Implementation
[0024] The following detailed description illustrates the specific implementation method: 1. American cockroach CYP9E2_2 Gene expression studies in different tissue sites 1.1 Experimental Materials and Equipment 1.1.11.1.1 Test insects and culture conditions The American cockroaches used in this experiment were all purchased from an American cockroach farm in Guangxi (a supplier of raw materials for Kangfuxin Liquid to Sichuan Good Doctor Pharmaceutical Group), and were an indoor artificially bred population. They were kept in fixed, breathable mesh containers, using egg cartons. The temperature was maintained at 28–30℃, and the humidity at approximately 65–75%, and they were kept in the dark. Both larvae and adults were fed rice, which was changed every two days.
[0025] 1.1.21.1.2 Instruments, Equipment and Related Reagents Table 1-1 Instruments and reagents involved in the experiment
[0026] 1.2 Experimental Content and Methods 1.2.1 Sample processing Adult American cockroaches were collected and subjected to a sublethal dose (LC). 50 American cockroaches were treated with a high-efficiency cyhalothrin solution of 875 mg / L. The cyhalothrin was prepared as a solvent using ultrapure water and sprayed onto the cockroaches using a spray bottle. Twenty cockroaches were treated per treatment, with three replicates. Afterward, the cockroaches were placed in a breathable plastic box. Two hours later, ten surviving cockroaches were collected and anesthetized with CO2. The muscles (MU), midgut (MI), and fat body (FA) were dissected. This process was repeated three times. Ten untreated cockroaches were also collected as a control (C) to obtain samples. All samples were promptly frozen in liquid nitrogen and then transferred to a -80°C freezer for later use.
[0027] 1.2.2 RNA Extraction Remove the frozen samples from 1.2.1 and extract total RNA from the American cockroaches using the TransZol Up Plus RNA Kit. Refer to the kit instructions and select the appropriate animal tissue RNA extraction method for the following steps: (1) Add a small amount of liquid nitrogen to the mortar and pre-cool it beforehand. Then take the sample out and put it into the mortar. Grind the sample thoroughly, adding liquid nitrogen continuously during the process. When the sample is ground into powder, transfer it into a 1.5 mL centrifuge tube.
[0028] (2) Add 1 mL Transzol Up and 0.2 mL RNA Extraction Agent to the sample, then pipette and mix repeatedly. Finally, vortex for at least 5 minutes until the color in the tube is uniform.
[0029] (3) Centrifuge at 1000×g and 4℃ for 15 min. After centrifugation, the centrifuge tube is divided into an upper layer (colorless aqueous phase), a middle layer (precipitate phase), and a lower layer (pink organic phase). At this time, the RNA is present in the colorless aqueous phase.
[0030] (4) Take 400-500 μL of aqueous phase and transfer it to a new centrifuge tube. At the same time, add anhydrous ethanol of the same volume as the aqueous phase, observe whether there is a precipitate, and gently invert to mix.
[0031] (5) Add the solution and precipitate from (4) to the centrifuge column, centrifuge at 12000×g for one minute at room temperature, and remove the effluent after centrifugation.
[0032] (6) Repeat step (5) once more.
[0033] (7) Prepare WB9 in advance (add 96 mL of anhydrous ethanol before use), add 500 μL of WB9 to the centrifuge column, centrifuge at 12000×g for one minute at room temperature, and discard the liquid in the tube after centrifugation.
[0034] (8) Repeat step (7) again.
[0035] (9) Centrifuge at 12000×g for two minutes at room temperature to completely remove residual ethanol.
[0036] (10) Take an RNase-free tube, put the centrifuge column into it, add 50-200 μL of RNase-free water in the center of the centrifuge column, and let it stand at room temperature for one minute.
[0037] (11) Centrifuge at 12000×g for one minute at room temperature to elute RNA.
[0038] (12) To ensure that more RNA is obtained, repeat steps (10) and (11) to perform a second elution.
[0039] (13) Transfer the RNA samples obtained from each part to a -80℃ freezer for storage.
[0040] (14) Take an appropriate amount of RNA, determine the concentration using an ultra-micro UV spectrophotometer, and determine the mass using an electrophoresis apparatus for agarose gel electrophoresis (buffer is 1×TAE).
[0041] 1.2.3 cDNA Synthesis RNA samples from each site described in section 1.2.2 were collected, and gDNA was removed and cDNA was synthesized using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit. The specific steps are as follows: (1) Configuration system: Table 1-2 Preparation system for synthesized cDNA
[0042] (2) Mix the above system by pipetting or gently shaking it manually.
[0043] If using Anchored Oligo (dT) or Gene-Specific Primers (GSP), incubate the product at 42°C for 15 minutes for qPCR. Alternatively, Random Primer (N9) can be used; incubate the product at 42°C for 15 minutes for qPCR as described above.
[0044] (3) After incubation, heat at 85°C for 5 seconds to inactivate EasyScript RT / RI and gDNA Remover to synthesize cDNA.
[0045] (4) Configure the system according to the table below (adjustments can be made according to the RNA concentration): Table 1-3 qPCR preparation system
[0046] 1.2.4 Determination by Real-Time Quantitative PCR CYP9E2_2 Expression of situation Prior to the experiment, Beijing Qingke Biotechnology Co., Ltd. was commissioned to design the relevant primers for the American cockroach. CYP9E2_2 The target gene is 18S rRNA, which is used as an internal reference gene.
[0047] Table 1-4 qPCR primer information
[0048] Centrifuge the prepared system from step 1.2.3 quickly in a handheld centrifuge (D1008E) to allow any remaining liquid on the walls of the eight-tube settling to the bottom, ensuring the liquid level is even in each tube. Then, use a quantitative PCR instrument (QuantStudio3) to set up the program for qPCR reaction: Table 1-5 qPCR instrument settings
[0049] After the reaction, observe the amplification curve analysis to determine whether nonspecific amplification occurred, and then use 2 -ΔΔCt The method calculates the relative gene expression level.
[0050] 2. Bioinformatics Analysis 2.1 Bioinformatics Analysis Content Choose American cockroaches CYP9E2_2For the genes, the nucleotide sequence was analyzed using the ORF Finder website, and information on gene sequences of other species was also searched. A phylogenetic tree was constructed using Mega software to determine the kinship between different species and the American cockroach. The physicochemical properties of the proteins were analyzed using ProtParam, and subcellular localization was performed using Cell-Ploc 2.0. Hydrophobicity prediction was performed using the ProScale online website to determine the protein's hydrophilicity. The protein signal peptide was analyzed using SignalP-5.0. Secondary structure was analyzed using the SPOMA online website, and the consistency with the tertiary structure predicted by Phyre2 was compared. Then, conserved domains and transmembrane domains were predicted using CD-Search and the TMHMH website, respectively. Glycosylation sites and phosphorylation sites were predicted using NetNGlyc-1.0 and NetPhos-3.1.
[0051] 2.2 Bioinformatics Analysis Software The software used in this analysis is shown in the table below: Table 2-1 Bioinformatics Analysis Software and Corresponding Websites
[0052] 3. Results and Discussion 3.1 Spatiotemporal expression pattern of the CYP450 gene in the American cockroach Both pairs of target genes used in this experiment were successfully amplified (e.g. Figure 1 ),illustrate CYP9E2_2 The gene was expressed in all American cockroaches after treatment with highly efficient cyhalothrin, indicating that... CYP9E2_2 The gene is involved in the detoxification process of the American cockroach against the highly effective cyhalothrin. Specifically: CYP9E2_2 The gene was expressed in muscle (MU), midgut (MI), fat body (FA), etc., and showed significant expression compared with the control group (C). The midgut showed the highest expression level. CYP9E2_2 Differences in the presence and distribution of genes likely indicate specific physiological functions. The midgut, as the most important organ in insects, is inextricably linked to their digestive and detoxification functions. It comes into direct contact with exogenous substances, undergoing digestion, absorption, and the development of resistance. In this experiment, the American cockroach likely primarily reacted with exogenous toxic substances through... CYP9E2_2 The gene is highly expressed in the midgut and metabolized to be excreted from the body, thereby achieving a detoxification effect.
[0053] 3.2 Bioinformatics Analysis Results 3.2.1 CYP9E2_2 Gene sequence analysis and phylogenetic tree construction Use the NCBI online website to search for American cockroaches. CYP9E2_2The gene's CDS region information was used to obtain its full length of 1581 bp, which was then input into the BLAST tool as *Periplaneta americana*. CYP9E2_2 By analyzing the amino acid CDS sequences and selecting appropriate species, the results were obtained for the American cockroach and its corresponding species. Cryptotermes secundus White pine new pine needle peak ( Neodiprion pinetum ), Pacific cockroach ( Diploptera punctata ), Timema tahoe Termites ( Coptotermes formosanus German cockroach ( Blattella germanica ), Gryllus longicercus Long-bearded sandfly ( Lutzomyia longipalpis ), bright desert locust ( Schistocerca nitens The sequence similarities were 72.49%, 47.50%, 53.13%, 66.10%, 70.63%, 70.78%, 47.01%, 49.91%, and 46.97%, respectively. Subsequently, the CDS sequences were downloaded and a phylogenetic tree was constructed using Mega software. The results are as follows: Figure 2 As shown: Comparing different species, it was found that the American cockroach and... C. secundus They are closely related, with white pine and new pine needle peaks ( N. pinetum ), Pacific cockroach ( D. punctata ), T. taho e is next, along with the German cockroach ( B. germanica ), subterranean termites ( C. formosanus ), G. longicercus Long-bearded sandfly ( L. longipalpis ), bright desert locust ( S. nitens The kinship is relatively distant.
[0054] 3.2.2 CYP9E2_2 Identification of protein information Will CYP9E2_2 The amino acid sequence was imported into ProtParam for analysis of the protein's physicochemical properties, and subcellular localization was performed using Cell-Ploc 2.0. The molecular formula was obtained as C1. 2742 H 4266 N 696 O 765 S 28， With a molecular weight of 60119.92 and a protein instability index of 35.97, it can be confirmed as a stable protein. Its theoretical isoelectric point is 7.17, indicating it is a weakly basic protein. The predicted half-life is 30 hours, and the N-terminus is methionine. The total number of negatively charged residues (Asp, Glu) is 63, and the total number of positively charged residues (Arg, Lys) is 63. Subcellular localization analysis suggests that this protein may interact with the endoplasmic reticulum and plasma membrane. Finally, the signal peptide (e.g., [missing information]) was predicted using the SignalP-5.0 tool. Figure 3 Analysis showed that the probability of the protein containing a signal peptide was 1.47%. CYP9E2_2 amino acid sequence: *
[0055] 3.2.3 CYP9E2_2 Protein hydrophilicity analysis Using the ProScale online website and combined with average hydrophobicity analysis (e.g.) Figure 4 American cockroach CYP9E2_2 In the protein, the phenylalanine at position 18 has the highest hydrophobicity score of 2.744, while the two valine residues at positions 448 and 449 have the lowest hydrophobicity scores of -2.656. Since the protein's average hydrophobicity coefficient is -0.170, it is classified as a hydrophilic protein.
[0056] 3.2.4 CYP9E2_2 Protein secondary and tertiary structure prediction Analysis of American cockroaches using the SPOMA online website CYP9E2_2 Protein secondary structure (e.g.) Figure 5The predicted results show that α-helices and β-sheets account for 42.97% and 4.75% respectively, while random coils and extended chains account for 37.45% and 14.83% respectively. Among them, α-helices and random coils account for a relatively high proportion and are the most important secondary structures. This also indicates that the protein has a certain degree of structural stability and structural diversity.
[0057] Analysis of American cockroaches using Phyre2 CYP9E2_2 Protein tertiary structure (e.g.) Figure 6 Further comparative analysis of the secondary structure confirms the above prediction results.
[0058] 3.2.5 CYP9E2_2 Prediction of conserved protein domains and transmembrane domains According to the analysis results of the CD-Search website (such as...) Figure 7 This indicates that the CYP6-like domain of the CYP450 protein family is contained in the American cockroach. CYP9E2_2 In proteins. And analysis of prediction results from the TMHMH official website (such as...) Figure 8 American cockroach CYP9E2_2 Proteins are a class of transmembrane proteins with one transmembrane domain located between positions 5 and 27 of the amino acid sequence, which is speculated to be involved in transmembrane transport.
[0059] 3.2.6 CYP9E2_2 Prediction of protein glycosylation sites and phosphorylation sites According to predictions from the DTU Health Tech website (such as...) Figures 9 - 10 ), CYP9E2_2 The protein has three phosphorylation modification sites, including serine, threonine, and tyrosine sites, with 14, 15, and 7 sites respectively. Among them, there may be 3 N-glycosylation modification sites with N-glycosylation potentials of 0.6458, 0.6147, and 0.5091 respectively. The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.
Claims
1. CYP9E2_2 Genes are characterized by: The nucleic acid sequence is shown in SEQ ID NO.
1.
2. The claim 1 CYP9E2_2 Application of genes in regulating resistance of American cockroaches to cyhalothrin.
3. As described in claim 2 CYP9E2_2 The application of genes in regulating resistance to cyhalothrin in American cockroaches is characterized by: Knockout CYP9E2_2 Genes that reduce the resistance of American cockroaches to cyhalothrin.
4. The encoding possesses the features described in claim 1 CYP9E2_2 The protein of a gene or its active fragment.
5. Containing the contents of claim 1 CYP9E2_2 Gene recombination vectors or host cells.
6. Targeted silencing as described in claim 1 CYP9E2_2 The gene's dsRNA, siRNA, or expression vector.
7. The claim 1 CYP9E2_2 Gene inhibitors.
8. Expressing or knocking out the expression described in claim 1 CYP9E2_2 Genetically modified insect cells, model organisms, or microorganisms.