Application of insect deubiquitinating enzyme BtUCH19 in pest control

Abstract

This invention discloses the application of the insect deubiquitinating enzyme BtUCH19 in the control of whiteflies, belonging to the field of plant protection. Using molecular cloning technology, the full-length sequence of the horizontally transferred gene BtUCH19 was successfully obtained, and its high expression in resistant whitefly populations was confirmed. Double-stranded RNA (dsBtUCH19) was synthesized using RNA interference (RNAi) technology, and after feeding treatment, the expression of the target gene was significantly inhibited, effectively improving the sensitivity of whiteflies to different neonicotinoid insecticides. This invention achieves highly efficient insecticidal activity while also considering ecological safety, possessing core advantages such as low toxicity, high efficacy, and high safety, providing important technical support for a green pest control system in the field.

Description

Technical Field

[0001] This invention belongs to the field of plant protection, specifically relating to the application of insect deubiquitinating enzymes in pest control, and more specifically, to the application of the fungal-derived horizontal transfer deubiquitinating enzyme BtUCH19 in the control of whiteflies, especially in the management of whitefly resistance to neonicotinoid insecticides. Background Technology

[0002] The tobacco whitefly (Bemisia tabaci) is a major global piercing-sucking agricultural pest belonging to the family Amycidae in the order Hemiptera. It has a wide host range, high reproductive capacity, and is highly migratory. Adults and nymphs feed on the phloem of plants, causing yellowing and wilting of leaves. They secrete honeydew, which induces sooty mold. Furthermore, they can transmit various plant viruses, causing devastating yield reductions or even crop failure in sensitive crops such as tomatoes. Currently, it has become one of the major threats to the development of the vegetable industry. In recent years, the core reason for the escalating damage caused by tobacco whiteflies is the spread of the invasive MED / Q cryptic species. This lineage exhibits extremely high resistance to traditional chemical pesticides, especially neonicotinoid insecticides, leading to a sharp decline in the efficacy of conventional pesticides in the field. This results in a vicious cycle of increased pesticide dosage and frequency, and continuously deteriorating efficacy. Pesticide resistance has become the core challenge restricting the effective control of tobacco whiteflies.

[0003] Existing research confirms that cytochrome P450 monooxygenases (CYPs)-mediated metabolic detoxification is the core mechanism by which whiteflies develop resistance to neonicotinoid insecticides. High expression of P450 genes such as CYP6CM1 and CYP4C64 can significantly enhance the whitefly's metabolic detoxification capacity against insecticides. Traditional resistance management and pesticide development largely revolve around the P450 main enzyme itself. However, the P450 enzyme system in insects is structurally complex, has high functional redundancy, and strong compensatory capabilities. Strategies targeting a single main enzyme are prone to cross-resistance and target failure, making it difficult to fundamentally solve the problem of resistance evolution.

[0004] The ubiquitin-proteasome system (UPS) is a core regulatory network in eukaryotes for maintaining protein homeostasis and signal transduction. Deubiquitinase (DUB), as a key "reverse switch" in the UPS, can precisely regulate the stability, subcellular localization, and activity of target proteins by specifically cleaving ubiquitin chains on substrate proteins, playing a crucial role in biological stress adaptation and metabolic regulation. Currently, the regulatory role of DUB in pesticide resistance in agricultural pests is not fully elucidated, and there are no specific control strategies for whiteflies targeting DUB.

[0005] Horizontal gene transfer (HGT) is one of the core characteristics of the whitefly's adaptive evolution. It allows the whitefly to acquire exogenous genetic material from cross-species organisms such as plants, bacteria, and fungi, and integrate it functionally, gaining adaptive advantages such as detoxifying plant secondary metabolites and enhancing symbiotic nutrition. Current research on whitefly HGT largely focuses on its host adaptability-related functions. Whether exogenous HGT genes participate in pesticide resistance regulation, how they interact with the endogenous P450 detoxification system, and the molecular mechanisms involved remain unclear, and no industrially applicable control technologies have been developed.

[0006] Therefore, identifying key DUB targets for whitefly resistance regulation, elucidating their molecular mechanisms of resistance, and developing green control technologies based on these targets are of significant theoretical and practical value for overcoming existing bottlenecks in whitefly resistance management, reducing the use of chemical pesticides, and promoting green and sustainable agricultural development. Summary of the Invention

[0007] To address the shortcomings of existing technologies, such as severe resistance of whiteflies to neonicotinoid insecticides, easy failure of traditional control targets, and difficulty in resistance management, the present invention aims to provide a novel molecular target for whitefly control—the horizontally transferred deubiquitinating enzyme gene BtUCH19. This invention clarifies its core regulatory role in whitefly resistance and provides gene interference agents and pest control methods based on this target. This achieves a strategic shift from "targeting the P450 master enzyme" to "targeting the upstream hub of resistance regulation," effectively improving the efficacy of insecticides against whiteflies and delaying resistance evolution.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides the application of the deubiquitinase gene BtUCH19 in pest control, wherein the nucleotide sequence of the BtUCH19 gene has the accession number Bta10703 in the whitefly genome database, and the pest is the whitefly, specifically the MED / Q cryptic species of the whitefly.

[0010] Furthermore, the application specifically involves: reducing the resistance of pests to pesticides, increasing the sensitivity of pests to pesticides, and / or enhancing the control effect of pesticides on pests by inhibiting the expression of the BtUCH19 gene or the activity of its encoded protein.

[0011] Furthermore, the inhibition of BtUCH19 gene expression or protein activity is achieved through RNA interference technology.

[0012] Furthermore, the insecticide is a neonicotinoid insecticide, preferably selected from at least one of imidacloprid, thiamethoxam, thiamethoxam, acetamiprid, acetamiprid, and thiamethoxam.

[0013] Secondly, the present invention provides a gene interference agent for pest control, the active ingredient of which is a double-stranded RNA targeting the deubiquitinase gene BtUCH19 of the whitefly. The double-stranded RNA is synthesized by RNA interference technology, and the gene interference agent is applied by feeding or spraying.

[0014] Furthermore, the double-stranded RNA is dsBtUCH19, and its nucleotide sequence is shown in SEQ ID NO.1.

[0015] Thirdly, the present invention provides the application of the above-mentioned gene interference agent in the preparation of products that inhibit or alleviate insecticide resistance in pests, wherein the pest is the whitefly.

[0016] Fourthly, the present invention provides a method for controlling whiteflies, specifically: feeding or spraying the whiteflies with the above-mentioned gene interfering agent to inhibit the expression of the BtUCH19 gene or the activity of its encoded protein, and then spraying an insecticide for pest control.

[0017] Furthermore, the feeding or spraying concentration of the gene interfering agent is 0.4-12 μg / μL, preferably 0.6-1.0 μg / μL; 6 hours after feeding or spraying the gene interfering agent, insecticide spraying treatment is performed.

[0018] Beneficial technical effects: This invention is the first to identify and confirm that the fungal-derived horizontal transfer gene BtUCH19 is a key regulatory factor in the resistance of whiteflies to neonicotinoid insecticides, and elucidates its novel resistance regulation molecular mechanism: the deubiquitinating enzyme encoded by BtUCH19 can specifically recognize and cleave the K63-linked polyubiquitin chain on the CYP4C64 protein, stabilize the abundance and catalytic activity of the CYP4C64 protein, and thus enhance the metabolic detoxification ability of whiteflies against neonicotinoid insecticides. This invention fills the research gap between horizontal gene transfer events and the regulation of insecticide resistance in whiteflies, and provides a new theoretical perspective for the study of insecticide resistance evolution.

[0019] This invention innovatively uses BtUCH19 as a novel molecular target for the control and resistance management of whiteflies. Unlike traditional neurotoxic targets and P450 master enzyme targets, this target, as an upstream hub for resistance regulation, has the technical advantage of "one-point intervention, systemic limitation". Targeted inhibition of this gene can weaken the P450-mediated metabolic detoxification ability from the root, and is less likely to induce target gene compensation and cross-resistance. It effectively breaks through the technical bottlenecks of existing synergists, such as poor specificity, insufficient field stability, and easy induction of adaptive evolution.

[0020] The gene-interfering agent targeting BtUCH19 developed in this invention can efficiently inhibit target gene expression through feeding or spraying, significantly increasing the sensitivity of resistant whitefly populations to neonicotinoid insecticides and greatly enhancing insecticide efficacy. This technical solution highly targets whiteflies with low off-target risk, achieving highly effective insecticidal control while significantly reducing the amount of chemical pesticides used in the field, extending the lifespan of existing insecticides, and reducing environmental pollution. It balances control efficacy, ecological safety, and field operability, providing important technical support and core patent layout for a green control system for whiteflies. Attached Figure Description

[0021] Figure 1 Transcriptome analysis of BtUCH19 in MED-resistant and susceptible populations of whiteflies.

[0022] Figure 2 The location of BtUCH19 in the MED strain of whitefly and the composition of the BtUCH19 protein domain, where a is the mRNA structure of BtUCH19 and b is the protein structure of BtUCH19.

[0023] Figure 3 Cross-species maximum likelihood phylogenetic tree analysis of the whitefly BtUCH19.

[0024] Figure 4 Phylogenetic tree analysis of deubiquitinating enzymes in whiteflies.

[0025] Figure 5 Identification of deubiquitination enzyme activity of BtUCH19 from the whitefly, where a is the BtUCH19 immunoprecipitation assay and b is the identification of the deubiquitination enzyme activity of BtUCH19 protein.

[0026] Figure 6 The differences in resistance multiples of resistant and sensitive whitefly populations under neonicotinoid insecticide (TMX, CLO) treatment, where a is a schematic diagram of TMX and CLO metabolism in whiteflies, and b is a graph showing the resistance multiples of resistant / sensitive populations to TMX and CLO.

[0027] Figure 7 The expression levels of BtUCH19 / BtUCH19 at the mRNA and protein levels in resistant whitefly populations were analyzed and quantified by qPCR and Western blot.

[0028] Figure 8 The expression levels of BtUCH19 / BtUCH19 in whiteflies after dsBtUCH19 interference were analyzed and quantified at the mRNA and protein levels using qPCR and Western blot.

[0029] Figure 9 Changes in the sensitivity of resistant populations to TMX and CLO after RNAi knockout of BtUCH19.

[0030] Figure 10 resistant population R #2 Immunoprecipitation analysis of BtUCH19 and CYP4C64 in China.

[0031] Figure 11 A schematic diagram of molecular docking between CYP4C64 and BtUCH19.

[0032] Figure 12 The yeast two-hybrid (Y2H) assay verified the in vitro interaction between BtUCH19 and CYP4C64.

[0033] Figure 13 The in vivo ubiquitination verification of CYP4C64 is shown in Figure a, where a is the Western blot (WB) detection of the ubiquitination degree of CYP4C64 after treatment with MG132 inhibitor, and a schematic diagram of the degradation of CYP4C64 by ubiquitination; and b is the Western blot identification of the K48 and K63 ubiquitination chains of CYP4C64 after treatment with MG132 inhibitor.

[0034] Figure 14 The expression of CYP4C64 and BtUCH19 proteins in prokaryotic Escherichia coli was detected by SDS-PAGE gel.

[0035] Figure 15GST-Pull-down analysis of CYP4C64 and BtUCH19 truncated mutants, where a) shows the binding of CYP4C64 truncated with normal BtUCH19 after GST-Pull-down analysis, and b) shows the binding of BtUCH19 truncated with normal CYP4C64 after GST-Pull-down analysis.

[0036] Figure 16 The ubiquitination level of CYP4C64.

[0037] Figure 17 Mutation analysis of CYP4C64 and BtUCH19 binding sites was performed. In case a, the predicted candidate ubiquitination sites of CYP4C64 were mutated to detect the degree of K63 modification of CYP4C64 after deubiquitination by normal BtUCH19. In case b, the candidate functional sites of BtUCH19 were mutated to detect the degree of K63 modification of CYP4C64 after deubiquitination by mutated BtUCH19. K48 was detected in the same way as in case a, and K48 was detected in the same way as in case b. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Example 1: Cloning of the whitefly horizontal transfer gene BtUCH19

[0042] Based on the Whitefly Genome Database and the NCBI database, the BtUCH19 gene in adult whiteflies was identified as a ubiquitin C-terminal hydrolase, with its gene sequence accession number in the Whitefly Genome Database (http: / / www.whiteflygenomics.org / cgi-bin / bta / blast.cgi). The full-length sequence of the BtUCH19 gene was obtained through molecular cloning.

[0043] First, total RNA was extracted from whiteflies and reverse transcribed to synthesize cDNA. The cloning primers for BtUCH19 were shown in Table 1, and PCR was performed. The PCR products were then recovered from the gel, ligated, and transformed. Positive single colonies were selected and sent to a company for sequencing to obtain the accurate sequence of the whitefly BtUCH19 gene. The PCR system is shown in Table 2, and the PCR reaction procedure is shown in Table 3.

[0044] Table 1. Primer information for BtUCH19 cloning

[0045] ;

[0046] Table 2. PCR reaction system

[0047] ;

[0048] Table 3. PCR reaction procedure

[0049] ;

[0050] Through Zheng et al. ZHENG H, XIE W, FU B, XIAO S, TAN X, JI Y, . . . ZHANG Y2021. Annual analysis of field-evolved insecticide resistance in Bemisiatabaci across China. Pest Manag. Sci. [J], 77: 2990-3001. DOI: 10.1002 / ps.6338.) The method of identifying B / Q populations of whiteflies was used, and all whiteflies were Q / MED populations (Mediterranean / MED). In pesticide-resistant whiteflies, the deubiquitinase (DUB) gene BtUCH19 (Identity: 98.23%, BTA013842.1, GenBank: XM_072302559.1, Figure 1 The value was significantly upregulated compared to the sensitive population.

[0051] Example 2: Analysis of ubiquitination enzyme activity of BtUCH19 deubiquitination enzyme

[0052] 1. To verify the water translocation event of BtUCH19 and demonstrate its deubiquitination enzyme activity, stepwise verification was performed using bioinformatics analysis and deubiquitination enzyme activity verification, respectively.

[0053] All deubiquitinating enzyme sequences were determined using the Whitefly Genomes website (http: / / www.whiteflygenomics.org / ftp / MED / ), gene locations were determined using BLAST from the NCBI website, and the protein molecular weight and isoelectric point of the deubiquitinating genes were predicted using ExPASy. A phylogenetic tree of 15,884 species was constructed using the RaxML randomized accelerated maximum likelihood method and homologous single-copy genes. Conserved domains of the BtUCH19 gene were predicted using the InterPro website (https: / / www.ebi.ac.uk / interpro / search / sequence / ) and the NCBI Cnoserved Domains search (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi). Possible deubiquitinating enzymes were screened using HMMER 3.1b from a protein family database. Amino acid sequences with more than 90% similarity within the same insect species were removed, and the outer ring of the phylogenetic tree was constructed using the remaining genes. The phylogenetic tree was constructed using MEGA11 (https: / / www.megasoftware.net / ) and then visualized using iTOL (https: / / itol.embl.de / ).

[0054] The mRNA sequence of BtUCH19 in the Q-type whitefly ( Figure 2 a) and protein ( Figure 2 b) Comparison and analysis results as follows Figure 2 As shown, BtUCH19 is 1176 bp in length and encodes 391 amino acids. The protein encoding BtUCH19 contains a zinc finger domain (zf-MYND), a typical characteristic domain of deubiquitinating enzyme-related proteins.

[0055] Figure 3 Phylogenetic analysis of BtUCH19 in different insect species showed that the BtUCH19 gene of the whitefly clustered with the UCH19 gene of the fungus, indicating that they have high sequence similarity and close evolutionary relationship, suggesting that the BtUCH19 gene of the whitefly originated from the fungus.

[0056] Functional and domain predictions of the BtUCH19 protein were performed, and the results showed that ( Figure 4 BtUCH19 belongs to the ubiquitin C-terminal hydrolase (UCH).

[0057] The specific degradation ability of BtUCH19 protein on substrates was systematically studied using an in vitro deubiquitinating enzyme activity assay. This invention also utilizes immunoprecipitation to analyze the insecticide-resistant whitefly population R... #2 The interactions between proteins were investigated, and rabbit immunoglobulin G was used as a specific negative control group to screen for possible target proteins.

[0058] 2. The extraction and detection of total protein from whiteflies shall be performed according to the following procedure:

[0059] Collect 200 adult whiteflies, flash-freeze in liquid nitrogen, add 1-2 steel balls, 400 μL of cell lysis buffer, and 4 μL of PMSF, and grind in a homogenizer 5-6 times. Centrifuge at 14000 rpm for 5 min at 4℃, and transfer the supernatant to a new 1.5 ml centrifuge tube. Quantify protein concentration using the BCA protein assay kit; the total protein quantification for each sample is 20 μg. Load 20 μL of the precast gel from a 4℃ freezer, and incubate at a constant voltage of 160 V for 60 min. After electrophoresis, perform membrane transfer. Place the PDVF membrane and filter paper in a plastic box, add 5 ml of activation buffer, and activate for 30 s. Transfer the target protein to the transfer buffer according to the marker size, in the following order: sponge, filter paper, gel block, PDVF membrane, filter paper, sponge. Secure the transfer with the clamps closed, place in the transfer tank, add an appropriate amount of transfer buffer, and place an ice pack in the transfer tank for cooling. Incubate at a constant current of 400 mA for 30 min. After transfer, wash the sample three times with 1×TBST, 8 min each time. After washing, add 5 ml of skim milk powder and incubate at 4°C for ≥3 hours at 75 rpm. After incubation, wash the sample five times with 1×TBST, 8 min each time. After washing, add 5 ml of skim milk powder and incubate overnight at 4°C with primary antibody at 75 rpm. The next day, wash the sample six times with 1×TBST, 8 min each time. After washing, add 5 ml of skim milk powder and incubate at 4°C with secondary antibody at room temperature for 45 min at 75 rpm. After secondary antibody incubation, wash the sample seven times with 1×TBST, 8 min each time, and then photograph with chemiluminescence.

[0060] Rabbit anti-BtUCH19 polyclonal antibody was prepared using a synthetic peptide (Qingke Biotechnology) as the antigen. The BtUCH19 peptide sequence is KRQFNQVADEPNPFD; β-actin was used as an internal control gene.

[0061] 3. Protein immunoprecipitation assay should be performed according to the following procedure:

[0062] Proteins were extracted using a total protein extraction kit in the presence of a 1× protease inhibitor to maintain protein integrity. The pre-cleaned lysis buffer was incubated overnight at 4°C with 5 μg of primary antibody against the target protein. Incubation with Protein A / G magnetic beads for 3 hours was performed to capture immune complexes. The protein was washed three times with IP lysis buffer, twice with high-salt buffer, and finally with PBS. The bound protein was eluted by incubation in 0.1 M glycine-HCl (pH 2.5) for 10 minutes with vigorous shaking. The eluent was collected by centrifugation at 12,000 × g, 4°C for 5 minutes. The eluent was neutralized with 1 M Tris-HCl (pH 8.0) at a 1:10 ratio to stabilize the target protein.

[0063] After separation by 10% SDS-PAGE gel electrophoresis, the enrichment of the target protein was detected by Western blot.

[0064] 4. The BtUCH19 deubiquitinase activity assay should be performed according to the following procedure:

[0065] For the assay of DUB protease activity, whitefly protein was extracted in the presence of a protease inhibitor (1:100 dilution) to maintain protein integrity. The sample was treated with pre-chilled lysis buffer containing 1 mM DTT and centrifuged at 10,000 × g for 15 minutes at 4°C. The supernatant was used for subsequent analysis. Immunoprecipitation was performed on the supernatant sample using an anti-BtUCH19 monoclonal antibody. After multiple washing and purification processes at 4°C, the BtUCH19 protease molecules with deubiquitination activity were enriched and isolated. After the experiment, the fluorescence signal intensity was measured using a multi-mode microplate reader with an excitation wavelength of 350 nm and an emission wavelength of 440 nm. Comparison with the data from the blank control group (PBS buffer) allowed for quantitative evaluation of the target substance's activity.

[0066] from Figure 5 As shown in section a, the deubiquitinating enzyme BtUCH19 was successfully extracted from live whiteflies. Under given conditions, this enzyme exhibits a significant trend of increasing activity over time. Figure 5 (b) Compared with the control group (PBS), the catalytic efficiency was higher.

[0067] Example 3: The whitefly BtUCH19 participates in the formation of two neonicotinoid agents.

[0068] To study the sensitivity of whitefly populations collected in the field to neonicotinoid insecticides, a feeding experiment was conducted using a self-developed bioassay device to screen and analyze the insecticide activity levels of adult whiteflies (15 males and 15 females) from different sources, i.e. insects from different tobacco field environments and different seasons.

[0069] Preparation of feed solutions for insecticides: Weigh out thiamethoxam and thiamethoxam technical grade (Yuanye, China), dissolve in acetone, and prepare a 10000 mg / L stock solution. According to the pre-designed protocol, the insecticide stock solution needs to be diluted multiple times to obtain a solution system with 6-7 concentration gradients. Cut a certain amount of Parafilm sealing film and stretch it so that the opening corresponds to the opening of the glass tube; add 60 μL of feed solutions with different insecticide concentrations to the sealing film, covering the liquid by stretching. Feed without added drugs serves as the control group, and feeds containing different concentrations of insecticides serve as the experimental groups. Each treatment is repeated 4 times independently. In the examples below, two different neonicotinoid insecticides are used; detailed information is shown in Table 4.

[0070] Table 4. Detailed information on neonicotinoid insecticides used in this experiment.

[0071] ;

[0072] a This indicates that the component descriptors of all active ingredients listed in this table are classified according to the Mechanism of Action (MoA) classification criteria established by the Insecticide Resistance Action Committee (IRAC).

[0073] 1. Bioassay analysis of adult insects:

[0074] Select 25-35 healthy adult whiteflies and place them in a specially designed glass container, sealing one end with sealing film. Use a fine needle to make two micro-holes in the film to ensure gas exchange. Place the device in a greenhouse at 25°C with a photoperiod of 14:10. After 48 hours, record the number of deaths and calculate the LC50 of the whiteflies using POLO Plus 2.0 (LeOra software, USA). 50。 Field trials showed that, compared with populations susceptible to whiteflies (S... #1 S #2 Compared to ), resistant populations (R) #1 R #2 The resistance to thiamethoxam and thiamethoxam was significantly higher, ranging from 144 to 319 times. Figure 6 As shown in a and b in the middle and in Table 5.

[0075] Table 5 shows the resistance multiples (RR) of whiteflies treated with two novel neonicotinoid insecticides. 50 value)

[0076] ;

[0077] a N, the number of test insects.

[0078] b FL, baseline confidence interval.

[0079] c x 2 , chi-square value.

[0080] d RR 50 Resistance factor = LC 50 (Test population) / LC 50 (The most sensitive population).

[0081] SE, standard error; R, resistant population; S, susceptible population.

[0082] 1. Quantitative analysis of the adult BtUCH19 gene:

[0083] The expression level of BtUCH19 in whiteflies resistant to allergies was analyzed by real-time quantitative qPCR. Based on the cDNA template obtained from the whitefly resistant population, qPCR primers for the BtUCH19 gene were synthesized (Table 6). The whitefly EF1α gene and ribosomal protein RPL29 gene were used as internal control genes. Analysis was performed using a QuantStudio 3 Real-time PCR System. -ΔΔCT The relative gene expression levels were calculated using the method shown in Table 7, and the procedure is shown in Table 8.

[0084] Table 6. Primers for whitefly qPCR

[0085] ;

[0086] Table 7. Quantitative Fluorescence System

[0087] ;

[0088] Table 8. Quantitative Fluorescence Procedure

[0089] ;

[0090] Compared to the susceptible population, the mRNA transcription level of the BtUCH19 gene in the resistant strain was approximately four times higher. Furthermore, Western blot analysis showed that the protein encoded by this gene was significantly higher in the resistant population (R...). #1 R #2 In more sensitive populations (S) #1 S #2 About 5 times higher Figure 7 The expression levels of BtUCH19 and BtUCH19 genes, both at the mRNA and protein levels, were significantly higher in the resistant population than in the susceptible population, indicating that high expression of these genes is significantly associated with resistance.

[0091] The expression of BtUCH19 and BtUCH19 was inhibited by RNAi using the double-stranded RNA of the BtUCH19 gene, as shown in SEQ ID NO. 1 (). The effect of this on the survival rate of the experimental population was assessed using insecticide bioassays. The synthesis method of dsBtUCH19 is as follows:

[0092] Insect exogenous dsRNA primers were designed using the Drosophila siRNA binding site website (http: / / www.flyrnai.org / cgi-bin / RNAi_find_primers.pl). Specific interference primers with a T7 linker (RNA polymerase promoter sequence) were designed.

[0093] The sequences of dsBtUCH19-F and dsBtUCH19-R are as follows:

[0094] dsBtUCH19-F: CCTGGTGCTCAGACTATGCCA (SEQ ID NO.2),

[0095] dsBtUCH19-R:ACAGGCTCGACAACTGCCT (SEQ ID NO.3);

[0096] dsRNA was synthesized using the Promega RNA double-strand synthesis kit.

[0097] Table 9. dsRNA synthesis system (10 μg DNA)

[0098] ;

[0099] Method of using dsBtUCH19: Synthesized dsBtUCH19 and dsBtUCH19 were diluted to 0.8 μg / μl with whitefly feeding solution (30% sucrose water) and fed to adult whiteflies. Live insects were collected after 24 h and 48 h for insecticide bioassay. dsEGFP was used as the experimental control group.

[0100] Changes in BtUCH19 expression levels in adult whiteflies after treatment with dsBtUCH19 ( Figure 8 ) and sensitivity determination to thiamethoxam and thiamethoxam after interference ( Figure 9 After treatment with dsBtUCH19, the mRNA and protein levels of neonicotinoid-resistant whiteflies decreased, and their sensitivity to neonicotinoids increased. After treatment with 0.8 μg / μL dsBtUCH19 for 6 h, the expression level of BtUCH19 in adult whiteflies decreased significantly by 42% (P<0.0001).

[0101] Figure 9 This indicates two resistant populations of whiteflies (R #1 R #2 ) and a sensitive population (S #1 S #2 The sensitivity of whiteflies to different neonicotinoid agents was determined after the mRNA level of BtUCH19 decreased. When resistant whiteflies were fed with feed solutions containing dsEGFP and dsBtUCH19 (1.0 / 0.6mM-TMX, 1.2 / 0.6mM-CLO), the mortality rate of whiteflies to the two neonicotinoid agents was significantly increased by 25-55% compared with the control group. This indicates that dsBtUCH19 significantly reduces the drug resistance level of whiteflies at both low and high concentrations, thereby increasing the sensitivity of whiteflies to insecticides.

[0102] Example 4: Resistant whiteflies are targeted to the key P450 detoxification enzyme gene via BtUCH19.

[0103] 1. Immunoprecipitation in vivo binding detection

[0104] In vivo binding assays using co-immunoprecipitation (Co-IP) confirmed the in vivo interaction between BtUCH19 and the key detoxification enzyme gene CYP4C64. Proteins were extracted from live whitefly homogenates and quantified using BCA (Biochemical Acetate Compound) assays. The mixture was incubated with BtUCH19 and CYP4C64 proteins, then magnetic beads were separated on a magnetic rack and washed five times with a washing buffer containing a low concentration of detergent. SDS-PAGE buffer was added to the magnetic beads, followed by heating to denature and elute the proteins. The resulting elution products were used for SDS-PAGE and Western blot analysis to detect the proteins.

[0105] The results showed that both BtUCH19 and CYP4C64 were detected. Figure 10 This indicates that BtUCH19 can directly interact with CYP4C64 in vivo.

[0106] 2. Molecular docking prediction

[0107] The molecular interactions between BtUCH19 and CYP4C64 were systematically analyzed using computational biology software. Simulated molecular docking of BtUCH19 and CYP4C64 was performed to predict and determine the domains and binding sites involved in their interaction.

[0108] Multiple ubiquitination sites of CYP4C64 were predicted using RUBI Version 1.0. 162 CYP4C64 164 CYP4C64 198 CYP4C64 236 and CYP4C64 281 ), and simulated molecular docking was performed. A molecular docking schematic diagram was constructed using PyMol, and certain amino acid residues of CYP4C64 (lysine / K) were observed. 236 ) through hydrogen bonds and salt bridges with specific amino acid residues (asparagine / N) of BtUCH19 286 Potential combination ( Figure 11 The interfacial area (Å) and ΔiG (solvation free energy gain, K) related to protein-protein interactions are also relevant. cal Table 10 provides detailed information on the number of chemical bonds (hydrogen bonds / NHB and salt bridges / NSB) and the number of chemical bonds (hydrogen bonds / NHB and salt bridges / NSB).

[0109] Table 10. Molecular docking parameters of CYP4C64 and BtUCH19

[0110] ;

[0111] Δ i G: Solvation free energy gain.

[0112] N: The number of chemical bonds.

[0113] HB Hydrogen bond.

[0114] SB Salt Bridge.

[0115] DS : Disulfide bond.

[0116] CSS: Complexation Importance Score.

[0117] Note: The parameters were simulated using PDBePISA (https: / / www.ebi.ac.uk / msd-srv / prot_int / ).

[0118] 3. Yeast two-hybrid (Y2H) in vitro binding detection

[0119] Yeast two-hybrid (Y2H) in vitro binding assays were performed, and based on molecular docking results, wild-type BtUCH19 and CYP4C64 vectors, as well as the mutant vector CYP4C64, were constructed. 162 CYP4C64 164 CYP4C64 198 CYP4C64 236 and CYP4C64 281 (The lysine / K residues at these sites were mutated to arginine / R, as shown in Table 11. These vectors were generated through in vitro experiments, and yeast two-hybrid (Y2H) experiments were performed to investigate the interactions between these constructs.)

[0120] Table 11. List of mutation sequences of BtUCH19 and CYP4C64 used in this patent

[0121] ;

[0122] Yeast strains were activated and competent yeast cells were prepared. These competent yeast cells can be used for subsequent experiments such as library construction or recombinant plasmid transformation. Subsequently, yeast cells were transformed with plasmids containing the relevant sequences of BtUCH19 and CYP4C64.

[0123] The system is shown in Table 12:

[0124] Table 12. Yeast Cell Transformation System

[0125] ;

[0126] Incubate at 30℃ for 30 min. Add 20 ml DMSO and incubate at 42℃ for 15 min, gently mixing every 5 min. Centrifuge at maximum speed for 15 s, aspirate the supernatant, add 1 μL YPD Plus Medium to resuspend, and incubate at 30℃ with shaking at 160 rpm for 1 h. Centrifuge at maximum speed for 15 s, aspirate the supernatant, add 1 ml 0.9% NaCl solution, and resuspend by pipetting. Spread 100 μL of the bacterial culture onto 100 mm selective solid medium of SD / -Trp (for pGBKT7-Y2HGold) and SD / eu (for pGADT7-Y187), and incubate at 30℃ inverted for 3-5 days until clones appear. Co-transformation products can be directly spread onto SD / -Leu-Trp, SD / -Leu-Trp-His, and SD / -Leu-Trp-His-Ade auxotrophic media to observe growth.

[0127] The results showed that when serine at positions 162 or 236 of the CYP4C64 protein was replaced, its inhibitory effect on yeast growth became very strong, especially in media with four deficiencies (SD, -Ade / adenine, -His / histidine, -Leu / leucine, -Trp / tryptophan). Figure 12 Therefore, the K amino acids at positions 162 and 236 of the CYP4C64 protein are binding sites and play an important role in ubiquitination.

[0128] Example 5: Key P450 deubiquitination enzyme gene in the resistant whitefly BtUCH19

[0129] 1. Ubiquitination inhibitor detection

[0130] To confirm the deubiquitination effect of BtUCH19 on CYP4C64, Western blot was used to detect CYP4C64 protein after treatment with MG132. MG132 is a highly efficient and specific inhibitor that can prevent the proteasome from selectively degrading target proteins via the ubiquitination pathway. The DMSO-treated group served as a control group. To elucidate the specific pathway by which CYP4C64 participates in the ubiquitin-proteasome system (UPS), K48 and K63 ubiquitin small molecule antibodies were used to detect CYP4C64 by Western blot.

[0131] The experimental results show that MG132 pretreatment significantly increased the relative expression level of CYP4C64 protein, by 15 times compared to the control group. Figure 13(a) When the ubiquitin-proteasome system is inhibited, the level of CYP4C64 protein increases. The results show that, compared to the blank control group, the knockout model exhibited nearly 5 times more K48 linkage modification events and nearly 10 times more K63 linkage modification events than baseline. Figure 13 (b) This indicates that K63 linkage may be the most important degradation regulation pathway. Studies have shown that both BtUCH1 and TRIM37 can specifically modify the CYP4C64 protein, causing it to be ubiquitinated and delivered into the protein degradation system. Furthermore, it was found that intracellular CYP4C64 is more inclined to use K63 linkage for precise regulation, thus allowing for fine-tuning of the organism's physiological functions.

[0132] 2. GST-Pulldown test to detect the binding affinity between BtUCH19 and CYP4C64

[0133] To thoroughly analyze the function of BtUCH19 in relation to CYP4C64, this paper established wild-type, truncated mutant, and site-directed mutant versions of BtUCH19 and CYP4C64 using various methods, and conducted in vitro expression and performance testing using different validation systems.

[0134] (1) Wild-type BtUCH19 and CYP4C64 were designed, and truncated mutants were constructed in pGEX-4T and pET-28a vectors, respectively, carrying GST or His tags (Table 13).

[0135] (2) Recombinant Pet28a-CYP4C64 and Pet28a-BtUCH19 proteins were induced by 0.5 mM IPTG at 16℃ for 48 hours.

[0136] (3) Recombinant pGEX-4T-1-CYP4C64 and pGEX-4T-1-BtUCH19 proteins were induced with 0.5 mM IPTG at 23°C for 24 hours.

[0137] (4) Add 100 µL of Glutathione Sepharose 4B to the Poly-Prep column. First, wash the column with about 100 mL of PBS to remove the preservation solution. Add 2 mL of protein supernatant with the GST tag and wash away any extraneous proteins with about 100 mL of PBS.

[0138] (5) The protein washing effect was detected by Coomassie Brilliant Blue G-250 staining method. After mixing 50 μL of PBS buffer and the elution buffer for contaminating proteins, if the solution showed no obvious blue color or only a slight light blue color, the washing step was complete. Approximately 50 μL of Glutathione Sepharose 4B material with a GST tag was taken and an SDS-PAGE sample was prepared according to the standard procedure for subsequent analysis.

[0139] (6) Take 2 mL of the supernatant containing different labeled proteins and wash repeatedly with about 100 mL of PBS solution to remove non-specific proteins. Coomassie Brilliant Blue assay.

[0140] (7) The supernatant was used to prepare SDS-PAGE protein samples for Western blot analysis. Empty vectors Pet28a and pGEX-4T-1 were used as controls. Before co-incubation with the expressed protein, its successful expression in E. coli was confirmed by SDS-PAGE and Coomassie brilliant blue staining. Figure 14 ).

[0141] Table 13. Vectors and gene fragments used in the experiment

[0142] ;

[0143] The prokaryotic expression of recombinant CYP4C64 and BtUCH19 proteins in *E. coli* was detected using SDS-PAGE and a pre-gel system, with empty vectors Pet28a and pGEX-4T-1 serving as controls. Coomassie brilliant blue staining revealed that both plasmids had successfully completed transcription and translation in host cells.

[0144] GST-Pull-down results showed that BtUCH19 interacts with CYP4C64 containing N-terminal residues 230-280, but does not interact with fragments 1-229 containing only the WxxxR motif (WxxxR) or fragments 281-510 of the conserved amino acid sequence PXXFXP (PxxFxP) near the carboxyl terminus. Figure 15 (a)

[0145] When BtUCH19 is truncated, it is found that CYP4C64 interacts with the N-terminal 100-290 region of BtUCH19, but does not interact with the 1-99 segment containing only the zinc finger domain or the 291-391 segment of BtUCH19. Figure 15 (b)

[0146] The relevant research results show that the interaction and binding between BtUCH19 and CYP4C64 are constituted by a specific functional region at their respective N-termini. This specific motif is formed by amino acids 100-290 of BtUCH19 and amino acids 230-280 of CYP4C64.

[0147] 2. In vitro expression verification of the binding sites of BtUCH19 and CYP4C64

[0148] To better understand the site-specific mechanism by which BtUCH19 regulates CYP4C64 function, this study used an in vitro protein expression system in S2 cells. The main focus was on establishing a co-expression model of TRIM37 and CYP4C64 genes in the S2 cell line to investigate the effect of plasmid-mediated gene transfer on CYP4C64 protein ubiquitination modification. Detailed studies were conducted using dynamic observations at different time points (24–72 h). The mutant vectors for BtUCH19 and CYP4C64 are shown in Table 14.

[0149] Table 14. Vectors and gene fragments used in the experiment

[0150] ;

[0151] 1. Passaging of S2 cells

[0152] (1) The initial seeding concentration during cell passage culture should not be less than 2 × 10⁻⁶. 6 / mL. Experimental results show that a faster growth rate can be obtained when S2 cells are cultured at high density, but excessive density will result in too many cells and inhibit growth.

[0153] (2) Take a cell density of 2×10 6 Add / mL of S2 cell suspension, mix thoroughly, and then add the appropriate volume of culture medium and shake.

[0154] (3) Transfer 6 mL of the mixed culture medium and divide it into three pre-washed 75 cm³ portions. 2 In petri dishes, add 8 mL of serum-containing culture medium to each fresh petri dish and gently shake to mix. Incubate at 28°C.

[0155] 2. Transfection of S2 cells

[0156] (1) Inoculating cells

[0157] The day before transfection, S2 cells were seeded in 6-well plates at a density of 3 × 10⁶ cells / well. 6 / mL—5×10 6 / mL per well, cultured in DMEM containing 10% serum for 18–22 hours.

[0158] (2) Transfection process

[0159] Prepare DNA dilution buffer (per well): Add 2 µg of plasmid DNA to 100 µL of serum-free DMEM medium and mix thoroughly. Prepare Lipo2000 dilution buffer (per well): In a separate 1.5 mL centrifuge tube, add 100 µL of serum-free DMEM medium and 4 µL of Lipo2000 (mix well before aspirating), mix well, and let stand for 5 min. Combine and mix, let stand for 20 min to obtain the DNA-Lipo2000 complex. Use a pipette to evenly add the DNA and Lipo2000 complex to the cell culture wells, and gently shake the 6-well plate to ensure thorough mixing. Then perform experiments for 24-72 hours.

[0160] Preliminary experimental results show that after CYP4C64 is transfected into S2 cells, the ubiquitination level of CYP4C64 reaches its peak within 12 hours, and K48 or K63 modification sites can be detected using specific antibodies. Co-immunoprecipitation showed that BtUCH19 cells carrying K162R and K236R mutations can inhibit the deubiquitination process of CYP4C64, with the most significant effect observed 12 hours post-transfection. Figure 16 ).

[0161] Single-gene mutation models were established for five possible ubiquitination binding sites in the CYP4C64 protein. The results showed that the K162R and K236R mutants were very insensitive to ubiquitination modification. Figure 17 (a). BtUCH19 is an important factor in CYP4C64 deubiquitination, and its function depends on the activity of proteolytic enzymes. Further experiments showed that the D286A mutant, which lacks catalytic function, cannot regulate deubiquitination in the S2 cell line. Figure 17 (b)

[0162] The figure shows that CYP4C64 ubiquitination via the K63 pathway is stronger than that via the K48 pathway, indicating that BtUCH19 increases the ubiquitination of CYP4C64 at the K236 site, stabilizing the K236 site and providing a crucial anchor point for BtUCH19 to recognize and process substrates. Similar validation can be performed on the K48 ubiquitination pathway. Figure 17 (c and d). The key site of BtUCH19 deubiquitinase activity is at D286 of the N-terminal fragment. Whether this region remains conserved determines whether BtUCH19 has an efficient degradation effect on the K63 type polyubiquitin chain of the target protein.

[0163] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of deubiquitinizing enzymes in pest control, characterized in that, The accession number of the nucleotide sequence of the gene BtUCH19 in the whitefly genome database is Bta10703, and the pest species is the whitefly.

2. The application according to claim 1, characterized in that, The application involves reducing insecticide resistance, increasing insecticide sensitivity, and / or increasing insecticide efficacy against pests by inhibiting BtUCH19 gene expression or protein activity.

3. The application according to claim 2, characterized in that, The inhibition is achieved through RNA interference.

4. The application according to claim 2, characterized in that, The insecticide is a neonicotinoid insecticide.

5. The application according to claim 4, characterized in that, The neonicotinoid insecticide is selected from at least one of imidacloprid, thiamethoxam, thiamethoxam, acetamiprid, acetamiprid, and thiamethoxam.

6. A gene-interfering agent for pest control, characterized in that, Its active ingredient is a double-stranded RNA that targets the deubiquitinase gene of the whitefly. The double-stranded RNA is synthesized using RNA interference technology, and the gene interference agent is used in the form of feeding or spraying.

7. The gene disruptor according to claim 6, characterized in that, The double-stranded RNA is dsBtUCH19, and its nucleotide sequence is shown in SEQ ID NO.

1.

8. The use of the gene disruptor according to any one of claims 6-7 in the preparation of products that inhibit or alleviate insecticide resistance.

9. A method for controlling pests, characterized in that, Feeding or spraying the pests with the gene-interfering agent as described in any one of claims 6-7 to inhibit the expression or activity of deubiquitinating enzymes, or spraying insecticides.

10. The method according to claim 9, characterized in that, The concentration of the gene-interfering agent used for feeding or spraying is 0.4-12 μg / μl, and the insecticide is sprayed 24h and 48h after feeding.

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