Insect adenosine triphosphate binding cassette superfamily transport protein and application thereof

Abstract

The invention relates to an insect adenosine triphosphate binding cassette superfamily transport protein and application thereof. The insect adenosine triphosphate binding cassette superfamily transporter is a homodimer, and each monomer has an adenosine triphosphate binding and hydrolysis structural domain, a transmembrane structural domain containing 6 transmembrane helixes and an extracellular structural domain; in a transmembrane structure domain of the protein structure, a spiral 1, a spiral 2 and a spiral 5 of each monomer form an arched substrate binding channel. Experiments show that the adenosine triphosphate binding cassette superfamily transporter and a pesticide micromolecule substrate are bound in the arched substrate binding channel and are discharged out of a body, so that drug resistance is generated, and the transport protein can be used for treating the pesticide micromolecule substrate. Therefore, the insect adenosine triphosphate binding cassette superfamily transport protein can be used as a target of insecticide resistance so as to design an efficient inhibitor compound, and can be used as a target spot for solving the insecticide resistance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an insect adenosine triphosphate binding cassette superfamily transporter protein and an application thereof. Background Art

[0002] Agricultural pests and diseases are important limiting factors for sustainable agricultural development. There are more than 730 common agricultural pests in my country, of which more than 20 are major agricultural pests. When pests reoccur, the crop yield loss caused by pests is as high as 30% or more. At present, chemical pesticide control is still the most important and widespread strategy for controlling most pests at this stage. However, recent studies have shown that many insects have evolved multiple resistance mechanisms and can develop resistance to a series of insecticides. The study of the resistance mechanism of agricultural pests has important theoretical significance for the rational use of pesticides and the effective control of insect pests. The adenosine triphosphate binding cassette superfamily transporter (ABC transporters) is one of the largest transmembrane protein families in organisms. It uses the hydrolysis of ATP to provide energy to promote the transmembrane transport of various substances. According to sequence similarity and conserved domains, the ABC transporter family can be divided into 8 subfamilies (ABCA-ABCH), and the number of members and functions of each subfamily are different. This type of protein is distributed in various organisms. Its main functions include transporting substances, signal transduction, cell surface receptors, and participating in intracellular DNA repair, transcription, and regulating gene expression. Recent studies have shown that mutation or overexpression of ABC transporters is not only closely related to arthropod resistance to chemical pesticides, but also plays a very important role in resistance to Bt toxins. ABCH subfamily transporters are ABC transporters unique to arthropods and can be used as a potential target for resistance screening. Elucidating the mechanism by which pests transport pesticide molecules to the outside of cells through ABCH transporters is of great significance for addressing the problem of insecticide resistance. Summary of the invention

[0003] In view of the needs of the prior art, the present invention provides a method for screening pesticide molecular substrates of ABCH transporter proteins based on ATPase activity, the cryo-electron microscopy structure of the red castaneum adenosine triphosphate binding cassette superfamily transporter ABCH and its complex with pesticide small molecules, and the structural information obtained through three-dimensional structural analysis and structural analysis can be used for pesticide design, which has broad application prospects in the field of pest control.

[0004] The technical solution of the present invention to solve the above technical problems is as follows:

[0005] The insect ATP-binding cassette superfamily transporter ABCH has a three-dimensional structure, in which the protein structure is a homodimer with C2 symmetry; each monomer of the protein structure has an ATP-binding and hydrolysis domain, a transmembrane domain containing 6 transmembrane helices and an extracellular domain, in which in the transmembrane domain of the protein structure, an "arched" substrate binding channel is formed by helix 1, helix 2, and helix 5 of each monomer.

[0006] The helix sequences of the "arched" substrate binding channel are: helix 1 sequence is NVGVMLFIFALPVMQVILFCLAI, helix 2 sequence is FTDFVAPGVILTIVFFLAVALTSSALIIER, and helix 5 sequence is ERNAIQLALGSFYPTLLL.

[0007] The insect ATP-binding cassette superfamily transporter ABCH, the insect is Tribolium castaneum Herbst; the amino acid sequence of the insect ATP-binding cassette superfamily transporter is shown in SEQ ID No.1.

[0008] The preparation method of insect adenosine triphosphate binding cassette superfamily transporter ABCH comprises the following steps:

[0009] 1) Expression and purification of ABCH protein

[0010] HEK293F cells were used to recombinantly express ABCH protein, and the collected cells expressing ABCH were disrupted, and high-purity ABCH protein was obtained by affinity chromatography and gel filtration chromatography;

[0011] 2) Cryo-EM sample preparation

[0012] (1) Concentrate high-purity ABCH protein to 5 mg / mL; (2) Glow discharge the R1.2 / 1.3, 300-mesh gold grid produced by Quantifoil for 30 seconds, with the glow discharge gas being H2 and O2; (3) Prepare the sample using FEI's Vitrobot Mark IV, and set the freezing sample preparation conditions as follows: blot-time: 3 s, wait-time: 5 s, drain-time: 0 s, blot-force: 3; (4) Take 3 μl of protein and add it to the surface of the grid. After the Vitrobot Mark IV completes the sample preparation procedure, the sample is quickly placed in liquid ethane and then stored in liquid nitrogen;

[0013] 3) Collect data and analyze the frozen samples under a cryo-electron microscope to obtain the three-dimensional structure of the protein ABCH.

[0014] The recombinant expression of ABCH protein in step 1) is performed by using the expression vector pcDNA3.1, transfecting the recombinant plasmid into HEK293F cells using the linear PEI transfection method, and expressing the protein under the conditions of 37° C., 150 rpm, and 5% CO 2 .

[0015] The method for preparing the high-purity ABCH protein in step 1) comprises the following steps:

[0016] (A) The collected HEK293F cells containing ABCH protein plasmid were suspended in pH 8.0, 20 mM Tris-HCl, 150 mM NaCl buffer, and the cells were broken by a high-pressure homogenizer, and then centrifuged at 13,000 rpm for 30 min. The supernatant was collected and ultracentrifuged at 150,000 g to collect the cell membrane. The cell membrane was dissolved in pH 8.0, 20 mM Tris-HCl, 150 mM NaCl, 1% Glyco-diosgenin for 1 h to fully dissolve the target protein on the cell membrane. The mixed components were ultracentrifuged at 150,000 g to collect the supernatant.

[0017] (B) The supernatant fraction is enriched by Strep affinity chromatography to enrich the target protein. After the supernatant passes through the strep affinity chromatography column, the impurities are washed with a buffer containing pH 8.0, 20 mM Tris-HCl, 150 mM NaCl, and 1% Glyco-diosgenin, and then the target protein is immediately eluted with a buffer containing pH 8.0, 20 mM Tris-HCl, 150 mM NaCl, 1% Glyco-diosgenin, and 50 mM biotin;

[0018] (C) After the protein was collected, it was concentrated to 500 μl and further purified using a Superose 6 gel filtration column to obtain a highly pure target protein.

[0019] Application of insect adenosine triphosphate-binding cassette superfamily transporter ABCH in screening small molecule pesticide substrates.

[0020] Application of insect adenosine triphosphate-binding cassette superfamily transporter ABCH as a target for insecticide resistance.

[0021] The use according to claim 8, comprising the steps of:

[0022] (a) Based on the principle of substrate activation of ATP hydrolysis activity, small molecule pesticide substrates of insect ATP-binding cassette superfamily transporter ABCH will be screened.

[0023] (b) Preparation of composite cryo-EM samples

[0024] (I) High-purity ABCH protein was concentrated to 5 mg / mL, and a small molecule pesticide substrate with a final concentration of 100 μM was added; (II) R1.2 / 1.3, 300 mesh gold grid of Quantifoil was treated with glow discharge for 30 seconds, and the glow discharge gas was H2 and O2; (III) Vitrobot Mark IV of FEI was used for sample preparation, and the freezing sample preparation conditions were set as follows: blot-time: 3s, wait-time: 5s, drain-time: 0s, blot-force: 3, (IV) 3 μl of protein was taken and added to the surface of the grid. After the VitrobotMark IV finished the sample preparation procedure, the sample was quickly placed in liquid ethane and then stored in liquid nitrogen;

[0025] (c) Collecting and analyzing data of frozen samples under a cryo-electron microscope to obtain the three-dimensional structure of protein ABCH and the binding site of small molecule pesticide substrates;

[0026] (d) The “arch” channel in the three-dimensional structure and the small molecule pesticide substrate binding site are used as targets to guide the design and development of resistance reversal agents targeting ABCH.

[0027] The small molecule pesticide substrate is fenoxycarb, and the binding sites are Arg404, Met405, Val411, Phe414, Phe608, Ser616, Ile707, Gln708, and Ala710.

[0028] The invention is based on the activation principle of substrate on ATP hydrolysis activity, and screens fenoxycarb as a potential pesticide small molecule substrate of ABCH by detecting the activation effects of 15 pesticide small molecules on the ATPase activity of ABCH.

[0029] The three-dimensional structure of the insect ATP-binding cassette superfamily transporter ABCH and the complex of ABCH and fenoxycarb was analyzed by cryo-electron microscopy technology. It is a three-dimensional structure, and the protein structure is a homodimer with C2 symmetry. Each monomer of the protein structure has an ATP binding and hydrolysis domain, a transmembrane domain containing 6 transmembrane helices and an extracellular domain. In the transmembrane domain of the protein structure, helix 1, helix 2, and helix 5 of each monomer form an "arched" substrate binding channel, and the helix sequence of the "arched" substrate binding channel is: NVGVMLFIFALPVMQVILFCLAI (helix 1), FTDFVAPGVILTIVFFLAVALTSSALIIER (helix 2), and ERNAIQLALGSFYPTLLL (helix 5). The two intracellular ends of the "arch" substrate binding channel are each bound to a molecule of fenoxycarb. The binding pocket is composed of Arg404, Met405, Val411, Phe414, Phe608, Ser616, Ile707, Gln708, and Ala710 ( Figure 5 ).

[0030] The present invention also evaluates the efflux activity of ABCH on fenoxycarb. After culturing cells transfected with ABCH or EGFP (control group) for 24 hours, fenoxycarb was added, and the cells were further cultured for 72 hours. The CCK8 kit was used to evaluate the cell viability. The results showed that the cells transfected with ABCH had a significant detoxification ability on fenoxycarb, indicating that ABCH can efflux fenoxycarb ( Figure 7 ).

[0031] The present invention takes the adenosine triphosphate-binding cassette superfamily transporter ABCH of Red Castanea as the object, and provides an efficient and universal recombinant protein expression and preparation method based on a two-step method of affinity chromatography and gel sieving, so as to obtain high-purity protein and prepare a cryo-electron microscopy sample, and analyze the complete structure of this protein by cryo-electron microscopy technology.

[0032] This application screened the activation principle of the substrate on the ATP hydrolysis activity and found that the fenoxycarb molecule may be the substrate of ABCH. The structure of the complex of ABCH and fenoxycarb was further analyzed, and it was found that fenoxycarb does exist in the substrate transport channel, indicating that fenoxycarb is indeed a substrate of ABCH. At the same time, through the efflux experiment based on cytotoxicity, it was shown that ABCH has the ability to detoxify fenoxycarb, that is, ABCH mediates the formation of insecticide resistance by effluxing fenoxycarb. The structure of the insect adenosine triphosphate binding cassette superfamily transporter ABCH has a narrow and long "arched" substrate binding channel. The pesticide substrate binds to the transporter ABCH through this binding channel and is excreted from the body through this channel. Therefore, this channel can be used as a target for insecticide resistance to guide the design and development of efficient inhibitors.

[0033] In addition, the application method provided by the present invention can be extended to all members of the ABC transporter family. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The figure is the purification result of ABCH recombinant protein, wherein the curve diagram is the elution curve of gel filtration chromatography, and the electrophoresis sample in SDS-PAGE is the protein sample after gel filtration purification.

[0035] Figure 2 It is the overall structure of the complex of ABCH and fenoxycarb. The whole is shown as a ribbon model, the fenoxycarb molecule is shown as a ball-and-stick model, and the box indicates the approximate position of the cell membrane.

[0036] Figure 3 This is the structural feature of the "arched" channel. Helix 1, helix 2, and helix 5 derived from two monomers form the "arched" channel, and the "arched" channel is displayed as a mesh model.

[0037] Figure 4 This is the binding mode of fenoxycarb. The protein cross-section shows the location of the "arch" channel. At the intracellular end, two fenoxycarb molecules are bound, and the fenoxycarb molecules are displayed as a ball-and-stick model.

[0038] Figure 5 The amino acid residues that form the fenoxycarb binding pocket.

[0039] Figure 6 These are the screening results for ABCH insecticide substrates.

[0040] Figure 7 This is the individual electron microscope structure of ABCH, and the overall structure appears as a ribbon model.

[0041] Figure 8 The vertical axis is the percentage of cell viability, where 1 is untransfected cells, 2 is untransfected cells plus fenoxycarb, 3 is EGFP-transfected cells, 4 is EGFP-transfected cells plus fenoxycarb, 5 is ABCH-transfected cells, and 6 is ABCH-transfected cells plus fenoxycarb. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0043] The reagents used in the examples are all commercially available.

[0044] The amino acid sequence of the Tribolium castaneum Herbst insect adenosine triphosphate binding cassette superfamily transporter ABCH selected by the present invention is shown in SEQ ID No.1.

[0045] Example 1: Preparation method of insect ATP-binding cassette superfamily transporter ABCH

[0046] 1. Construction of ABCH expression vector

[0047] (1) A HindIII restriction site sequence and a Twin-strep purification tag were inserted into the nitrogen end of the ABCH protein, and an XhoI restriction site sequence was inserted into the carbon end. Codon optimization was performed for HEK293 cells to synthesize the full-length protein gene sequence, as shown in SEQ ID No. 2.

[0048] (2) The full-length gene sequence and pcDNA3.1 were digested with HindIII and XhoI restriction enzymes and ligated with T4 DNA ligase to obtain a recombinant expression vector. Double restriction enzyme digestion and plasmid sequencing confirmed that the expression vector was successfully constructed.

[0049] (3) The recombinant plasmid was transformed into Escherichia coli DH5α competent cells using the chemical transformation method to obtain a cloning strain.

[0050] 2. Expression of ABCH protein

[0051] (1) Plasmid extraction

[0052] The cloned strain was cultured in LB medium at 37°C overnight, and a high-concentration, high-purity recombinant plasmid was extracted using a plasmid extraction kit (Tiangen Biotechnology) and stored at -20°C for later use.

[0053] (2) Recovery and passaging of HEK293F cells

[0054] HEK293F cells were taken out of liquid nitrogen and quickly thawed in a 37°C water bath for about 2 minutes. After thawing, the cells were immediately transferred to preheated culture medium and cultured at 150 rpm, 37°C, and 5% carbon dioxide. When the cell density reached 3×10 6 After that, the cells were subcultured, and the initial density of the subculture was controlled at 0.2-0.3×10 6 .

[0055] (3) HEK293F cell transfection

[0056] HEK293F cells were transfected using a linear PEI cationic polymer transfection method. The transfection method is as follows:

[0057] Before transfection, the cell density was diluted to 1 × 10 6 Continue to culture for 3-4h or overnight, and control the density at 2×10 6 Within. Add the plasmid to 10ml preheated fresh culture medium at a volume of 2ug / ml culture medium, vortex to mix, take 3 times the amount of PEI solution, add it to the culture medium containing the plasmid, slowly add it dropwise, vortex and shake while adding, vortex and shake vigorously for 10s after adding, let it stand for 30min to form PEI-DNA complex. After standing, slowly add the PEI-DNA complex culture medium solution to the cultured cells, shake the shake bottle while adding to mix it evenly, then culture it at 150rpm, 37℃, 5% carbon dioxide for 2 days, and collect the cells by centrifugation at 1000g for 5min.

[0058] 3. Purification of ABCH Protein

[0059] (1) The collected HEK293F cells were suspended in 20 Mm Tris-HCl (pH 8.0), 150 Mm NaCl buffer, and the cells were broken by a high-pressure homogenizer. Then, the cells were centrifuged at 13,000 rpm for 30 min, and the supernatant was collected. The cell membranes were collected by ultracentrifugation at 150,000 g. The cell membranes were suspended in 20 Mm Tris-HCl (pH 8.0), 150 Mm NaCl, 1%

[0060] The mixture was dissolved under the condition of Glyco-diosgenin for 1 hour to fully dissolve the target protein on the cell membrane. The mixed components were ultracentrifuged at 150,000 g and the supernatant was collected.

[0061] (2) The supernatant fraction was enriched for the target protein by Strep affinity chromatography. After the supernatant passed through the strep affinity chromatography column, the impurities were washed with a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% Glyco-diosgenin, and then the target protein was immediately eluted with a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Glyco-diosgenin, and 50 mM biotin.

[0062] (3) After the protein was collected, it was concentrated to 500 μl and further purified using a Superose 6 gel filtration column to obtain a high-purity target protein. Through the above purification steps, a high-purity ABCH protein was obtained (see the attached results). Figure 1 ), which can be used for the preparation of cryo-EM samples.

[0063] Example 2: Screening of pesticide small molecule substrates for insect ATP-binding cassette superfamily transporter ABCH

[0064] The ATP hydrolysis activity of ABCH is determined using the pyruvate-coupled assay. The assay is based on a reaction system that regenerates the hydrolyzed ATP while producing pyruvate. After each cycle of ATP hydrolysis catalyzed by ABCH, a regeneration system consisting of phosphoenolpyruvate (PEP) and pyruvate kinase (PK) converts one molecule of PEP into pyruvate, which in turn reconverts ADP into ATP. Pyruvate reacts with 2,4-dinitrophenylhydrazine to produce acetone-2,4-dinitrophenylhydrazone, which can be quantified by measuring the absorbance at 450 nm, i.e., the amount of pyruvate produced is equal to the amount of ATP consumed.

[0065] The ATPase activity of purified ABCH in detergent was determined at 30°C. First, ABCH was incubated with different pesticide small molecules for 10 minutes, and the ABCH sample without any small molecules was used as a control. The incubation system contained 50mM Tris (pH 8.0), 2mM MgCl2, 150mM KCl, 1mM DTT, and 0.03% Glyco-diosgenin. Subsequently, 5mM PEP, 20U / ml pyruvate kinase, and 2mM ATP were added to the system. After 30 minutes, the reaction was stopped by adding 100μL of a solution containing 0.36mM 2,4-dinitrophenylhydrazine and 0.4M HCl, and incubated at 37°C for 15 minutes. Finally, 100μL of 2.5M NaOH and 0.1M EDTA were added to each reaction, and the absorbance at 450nm was recorded using a 96-well plate, and the ATPase enzyme activity was calculated using the pyruvate standard curve. By comparing the activation effects of different pesticide small molecules on ABCH ATPase activity, we can explore possible pesticide small molecule substrates of ABCH (screening results are shown in Figure 6 ).

[0066] The results of ATPase-based insecticide substrate screening showed that fenoxycarb could significantly stimulate the ATPase activity of ABCH, indicating that fenoxycarb is a potential insecticide substrate of ABCH.

[0067] Example 3: Preparation of frozen samples of Tribolium castaneum ABCH and its complex with fenoxycarb

[0068] (1) In 50 mM Tris (pH 8.0), 150 mM NaCl, 2 mM DTT, 2 mM Mg 2+ , 0.05% Glyco-diosgenin was used to obtain high-purity ABCH protein, which was concentrated to 5 mg / mL;

[0069] (2) A Quantifoil R1.2 / 1.3, 300 mesh gold grid was glow-discharged for 30 seconds using H2 and O2 as the glow discharge gas.

[0070] (3) Use FEI's Vitrobot Mark IV for sample preparation, and set the freezing sample preparation conditions (blot-time: 3 s, wait-time: 5 s, drain-time: 0 s, blot-force: 3).

[0071] (4) Take 3 μl of protein and add it to the surface of the grid. After the Vitrobot Mark IV completes the sample preparation process, the sample is quickly placed in liquid ethane and then stored in liquid nitrogen.

[0072] The frozen samples of the complex of ABCH and fenoxycarb were prepared in the same manner, except that in step (1), fenoxycarb (dissolved in DMSO) was added at a final concentration of 100 μM after protein concentration, and the samples were prepared in the same manner after incubation for 30 minutes.

[0073] Example 4: Structural analysis of Tribolium castaneum ABCH alone and its complex with fenoxycarb

[0074] The cryo-samples were first screened at 200 kV, and then data were collected at 300 kV. The electron microscopes used for data collection were FEI's Titan Krios and Gatan's K2 summit direct electron detection camera. Data were collected automatically using Serial EM software in low-dose mode. The magnification was set to 22,500 times, corresponding to a pixel size of The total electron dose is The defocus value range is set to -1 to -2 μm.

[0075] For the ABCH sample alone, a total of 5954 single particle images were collected, and the image data were processed by cryoSPARC software to obtain a resolution of For the ABCH and fenoxycarb complex sample, a total of 5243 single particle images were collected, and the image data were processed by cryoSPARC software to obtain a resolution of The electron density of the protein was then determined using PHENIX and Coot to build and fine-tune the structure based on the protein sequence, and the structure was evaluated using PDB Validation Service (see Table 1 for structural parameters).

[0076] Table 1 Cryo-EM data collection and structure correction data statistics

[0077]

[0078] The ABCH structure obtained by analysis is the first H subfamily adenosine triphosphate binding cassette superfamily transporter. The protein structure is a homodimer, and each monomer contains a nucleotide binding domain (NBD) located inside the cell, an extracellular domain (ECD) located outside the cell, and a transmembrane domain (TMD) containing 6 transmembrane helices. ( Figure 2 , Figure 7 ), the TMD domain of the dimeric protein forms an "arch" channel, which is formed by helix 1, helix 2 and helix 5 of the TMD domain ( Figure 3 ), the helical structure sequences involved are NVGVMLFIFALPVMQVILFCLAI (helix 1), FTDFVAPGVILTIVFFLAVALTSSALIIER (helix 2), ERNAIQLALGSFYPTLLL (helix 5). Two fenoxycarb binding pockets are formed at the two ends of the "arch" channel close to the intracellular side ( Figure 4 ), the binding pocket is composed of Arg404, Met405, Val411, Phe414, Phe608, Ser616, Ile707, Gln708, Ala710. ( Figure 5 ), the structural details obtained can be applied to the design of structure-based resistance reversal agents.

[0079] Example 5: Detoxification experiment of ABCH on fenoxycarb

[0080] In this experiment, HEK293F cell line was used to evaluate the detoxification ability of ABCH. First, HEK293 cells were cultured in culture medium to 1.5×10 6 The density was then determined by transfecting HEK293F cells with ABCH and EGFP control plasmids using linear PEI 40000 transfection reagent, and culturing was continued for 24 hours to allow the transfected proteins to be expressed. Subsequently, 50 nM fenoxycarb was added, and culturing was continued for 72 hours. The cell activity was detected using a CCK8 kit.

[0081] The results showed that cells transfected with ABCH had a significant detoxification ability to fenoxycarb, indicating that ABCH could excrete fenoxycarb ( Figure 8 )

[0082] The detoxification effect of ABCH on fenoxycarb, combined with the structural information of the complex of ABCH and fenoxycarb, can prove that ABCH mediates the development of fenoxycarb resistance by recruiting fenoxycarb molecules into the "arch" substrate transport channel to achieve the efflux of fenoxycarb. Therefore, the "arch" channel and the fenoxycarb binding site can be used as targets to guide the design and development of resistance reversal agents targeting ABCH.

Claims

1. The insect ATP-binding cassette superfamily transporter ABCH has a three-dimensional structure, in which the protein structure is a homodimer with C2 symmetry; each monomer of the protein structure has an ATP-binding and hydrolysis domain, a transmembrane domain containing 6 transmembrane helices and an extracellular domain, wherein in the transmembrane domain of the protein structure, a "arched" substrate binding channel is formed by helix 1, helix 2, and helix 5 of each monomer.

2. The insect ATP-binding cassette superfamily transporter ABCH according to claim 1, wherein the insect is Tribolium castaneum Herbst; the amino acid sequence of the insect ATP-binding cassette superfamily transporter is shown in SEQ ID No.

1.

3. According to the insect adenosine triphosphate binding cassette superfamily transporter ABCH according to claim 2, the helix sequence of the "arched" substrate binding channel is: the helix 1 sequence is NVGVMLFIFALPVMQVILFCLAI, the helix 2 sequence is FTDFVAPGVILTIVFFLAVALTSSALIIER, and the helix 5 sequence is ERNAIQLALGSFYPTLLL.

4. The method for preparing the insect adenosine triphosphate binding cassette superfamily transporter ABCH according to any one of claims 1 to 3, comprising the following steps: 1) Expression and purification of ABCH protein HEK293F cells were used to recombinantly express ABCH protein, and the collected cells expressing ABCH were disrupted, and high-purity ABCH protein was obtained by affinity chromatography and gel filtration chromatography; 2) Cryo-EM sample preparation (1) Concentrate high-purity ABCH protein to 5 mg / mL; (2) Glow discharge the R1.2 / 1.3, 300-mesh gold grid produced by Quantifoil for 30 seconds, with H2 and O2 as the glow discharge gas; (3) Prepare the sample using FEI's Vitrobot Mark IV, and set the freezing sample preparation conditions as follows: blot-time: 3s, wait-time: 5s, drain-time: 0s, blot-force: 3; (4) Take 3 μl of protein and add it to the surface of the grid. After the Vitrobot Mark IV completes the sample preparation procedure, the sample is quickly placed in liquid ethane and then stored in liquid nitrogen; 3) Collect data and analyze the frozen samples under a cryo-electron microscope to obtain the three-dimensional structure of the protein ABCH.

5. The preparation method according to claim 4, wherein the recombinant expression of ABCH protein in step 1) is performed by using an expression vector pcDNA3.1, transfecting the recombinant plasmid into HEK293F cells using a linear PEI transfection method, and expressing the protein at 37°C, 150rpm, and 5% CO2.

6. The preparation method according to claim 4, wherein the preparation method of the high-purity ABCH protein in step 1) comprises the following steps: (A) The collected HEK293F cells containing ABCH protein plasmid were suspended in pH 8.0, 20 mM Tris-HCl, 150 mM NaCl buffer, and the cells were broken by a high-pressure homogenizer, and then centrifuged at 13,000 rpm for 30 min. The supernatant was collected and ultracentrifuged at 150,000 g to collect the cell membrane. The cell membrane was dissolved in pH 8.0, 20 mM Tris-HCl, 150 mM NaCl, 1% Glyco-diosgenin for 1 h to fully dissolve the target protein on the cell membrane. The mixed components were ultracentrifuged at 150,000 g to collect the supernatant. (B) The supernatant fraction is enriched by Strep affinity chromatography to enrich the target protein. After the supernatant passes through the strep affinity chromatography column, the impurities are washed with a buffer containing pH 8.0, 20 mM Tris-HCl, 150 mM NaCl, and 1% Glyco-diosgenin, and then the target protein is immediately eluted with a buffer containing pH 8.0, 20 mM Tris-HCl, 150 mM NaCl, 1% Glyco-diosgenin, and 50 mM biotin; (C) After the protein was collected, it was concentrated to 500 μl and further purified using a Superose 6 gel filtration column to obtain a highly pure target protein.

7. Application of insect adenosine triphosphate-binding cassette superfamily transporter ABCH in screening small molecule pesticide substrates.

8. Use of the insect adenosine triphosphate binding cassette superfamily transporter ABCH according to any one of claims 1 to 3 as an insecticide resistance target.

9. The use according to claim 8, comprising the following steps: (a) Based on the principle of substrate activation of ATP hydrolysis activity, small molecule pesticide substrates of insect ATP-binding cassette superfamily transporter ABCH will be screened. (b) Preparation of composite cryo-EM samples (I) High-purity ABCH protein was concentrated to 5 mg / mL, and a small molecule pesticide substrate was added at a final concentration of 100 μM; (II) R1.2 / 1.3, 300 mesh gold grid of Quantifoil was glow-discharged for 30 seconds, and the glow discharge gas was H2 and O2; (III) Vitrobot MarkIV of FEI was used for sample preparation, and the freezing sample preparation conditions were set as follows: blot-time: 3s, wait-time: 5s, drain-time: 0s, blot-force: 3; (IV) 3 μl of protein was taken and added to the surface of the grid. After the Vitrobot Mark IV finished the sample preparation procedure, the sample was quickly placed in liquid ethane and then stored in liquid nitrogen; (c) Collecting and analyzing data of frozen samples under a cryo-electron microscope to obtain the three-dimensional structure of protein ABCH and the binding site of small molecule pesticide substrates; (d) The "arch" channel in the three-dimensional structure and the small molecule pesticide substrate binding site are used as targets to guide the design and development of resistance reversal agents targeting ABCH.

10. The use according to claim 9, wherein the small molecule pesticide substrate is fenoxycarb, and the binding sites are Arg404, Met405, Val411, Phe414, Phe608, Ser616, Ile707, Gln708, and Ala710.

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