Transporter ABCH and LMNG compound structure and application of detergent molecule LMNG as insecticide

Abstract

The invention relates to a transporter ABCH and LMNG compound structure and application of a detergent molecule LMNG as an insecticide, and the LMNG has obvious inhibitory activity on ABCH and obvious insecticidal activity on larvae of tribolium castaneum and plutella xylostella. The invention relates to an insect adenosine triphosphate binding cassette superfamily transporter ABCH and LMNG compound structure which is a three-dimensional structure, and the protein structure is a homodimer and has C2 symmetry; each monomer of the protein structure has an adenosine triphosphate binding and hydrolysis structural domain, a transmembrane structural domain containing 6 transmembrane helixes and an extracellular structural domain; and the adenosine triphosphate binding and hydrolysis structural domain is connected with the transmembrane structural domain through a connecting helix.

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 inhibitor and application thereof. Background Art

[0002] With the development of global agricultural intensification, pest resistance has become a major challenge in the use of pesticides. In recent years, pest resistance to chemical pesticides has continued to increase, resulting in reduced crop yields, rising production costs, and intensified environmental pollution. Therefore, the development of new pesticides and inhibitors targeting new targets is of great significance for dealing with agricultural pest resistance, which can not only avoid the environmental pressure caused by increasing the amount of pesticide application, but also accurately and reasonably achieve efficient insecticide control. ABC transporters are key players in the formation of pesticide resistance. Elucidating their substrate recognition and transport mechanisms is of great significance for the development and design of resistance reversal agents. The ABC transporter family is divided into 8 subfamilies (ABCA-ABCH), among which the ABCH subfamily transporter is an ABC transporter unique to arthropods. Studies have shown that ABCH subfamily transporters, as lipid transport proteins, play a key role in building the lipid barrier of insect cuticles. Silencing the expression of ABHC genes in red castaneum, East Asian migratory locust, and diamondback moth by RNA interference can lead to significant lethality, among which silencing the ABCH gene of red castaneum can lead to 100% lethality. Therefore, ABCH subfamily members can be used as potential insecticidal targets, and the screening and design of inhibitors targeting ABCH provides new ideas for the development of new insecticides. Summary of the invention

[0003] In view of the needs of the prior art, the present invention provides a detergent molecule (Lauryl maltose neopentyl glycol, LMNG) that inhibits the ATP hydrolysis activity of the red castaneum adenosine triphosphate binding cassette superfamily transporter ABCH, the cryo-electron microscopy structure of red castaneum ABCH and its complex with LMNG, and the binding site information obtained through three-dimensional structure analysis and structural analysis can be used for insecticide 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 cryo-EM structure of the insect ATP-binding cassette superfamily transporter ABCH and LMNG complex is 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 (NBD), a transmembrane domain (TMD) containing 6 transmembrane helices, and an extracellular domain (ECD), and the ATP-binding and hydrolysis domain is connected to the transmembrane domain through a connecting helix (CnH). Two LMNG binding sites are formed at the interface between the ATP-binding and hydrolysis domains and the transmembrane domains of the two monomers, which contain amino acid residues on the ATP binding loop, connecting helix CnH, transmembrane helix 1 (TM1), transmembrane helix 2 (TM2), transmembrane helix 3 (TM3), and transmembrane helix 5 (TM5).

[0006] The binding site substrate binding site is: E155, K197 amino acid residues from the ATP binding loop, N401, R404 amino acid residues from the connecting helix, F414 amino acid residue from the transmembrane helix TM1, A612, L613 amino acid residues from the transmembrane helix TM2, H644, Q648 amino acid residues from the transmembrane helix TM3, and L702, R704, N705, Q708 amino acid residues from the transmembrane helix TM5.

[0007] The insect adenosine triphosphate binding cassette superfamily transporter ABCH inhibitor is lauryl maltoseneopentyl glycol, referred to as LMNG, which is a detergent molecule.

[0008] 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.

[0009] Application of insect adenosine triphosphate-binding cassette superfamily transporter ABCH as insecticide target.

[0010] The application comprises the following steps:

[0011] (a) Based on the ATP hydrolysis experiment, the effects of different detergents on the ATP hydrolysis activity of insect ATP-binding cassette superfamily transporter ABCH were screened to obtain detergents that inhibit the activity of insect ATP-binding cassette superfamily transporter ABCH;

[0012] (b) Preparation of composite cryo-EM samples

[0013] (I) The high-purity ABCH protein obtained in a detergent environment that inhibits the activity of insect ATP-binding cassette superfamily transporter ABCH was concentrated to 5 mg / mL; (II) The R1.2 / 1.3, 300-mesh gold grid produced by Quantifoil was glow-discharged for 30 seconds, and the glow-discharge gas was H2 and O2; (III) The sample was prepared using FEI's Vitrobot Mark IV, and the freezing sample preparation conditions were set as follows: blot-time: 3s, wait-time: 5s, drain-time: 0s, blot-force: 3; (IV) 3.5 μL of protein was taken and added to the surface of the grid. After the Vitrobot Mark IV completed the sample preparation procedure, the sample was quickly placed in liquid ethane and then stored in liquid nitrogen;

[0014] (c) The frozen samples were collected and analyzed under a cryo-electron microscope to obtain the three-dimensional structure of the protein ABCH and the detergent binding site that inhibits the activity of the insect ATP-binding cassette superfamily transporter ABCH;

[0015] (d) The detergent binding site that inhibits the activity of insect adenosine triphosphate-binding cassette superfamily transporter ABCH in the three-dimensional structure is used as a target to guide the design and development of insecticides targeting ABCH.

[0016] In the application, the detergent molecule is LMNG, and the binding sites are E155 and K197 amino acid residues from the ATP binding loop, N401 and R404 amino acid residues from the connecting helix, F414 amino acid residue from the transmembrane helix TM1, A612 and L613 amino acid residues from the transmembrane helix TM2, H644 and Q648 amino acid residues from the transmembrane helix TM3, and L702, R704, N705, and Q708 amino acid residues from the transmembrane helix TM5.

[0017] In the application, the method for preparing high-purity ABCH protein in step (I) is: using HEK293F cells to recombinantly express ABCH protein, crushing the collected ABCH-expressing cells, and obtaining high-purity ABCH protein by affinity chromatography and gel filtration chromatography.

[0018] The affinity chromatography and gel filtration chromatography comprise the following steps:

[0019] (A) The collected HEK293F cells containing ABCH protein particles were suspended in pH 8.0, 20mM Tris-HCl, 150mM NaCl buffer, and the cells were broken by a high-pressure homogenizer, and then centrifuged at 13000rpm for 30min. The supernatant was collected and ultracentrifuged at 150000×g to collect the cell membrane. The cell membrane was dissolved in pH8.0, 20mMTris-HCl, 150mM NaCl, 1% LMNG for 1h to fully dissolve the target protein on the cell membrane. The mixed components were ultracentrifuged at 150000×g to collect the supernatant.

[0020] (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;

[0021] (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.

[0022] The recombinant expression of ABCH protein in step (I) 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 .

[0023] Application of LMNG as an inhibitor of insect adenosine triphosphate-binding cassette superfamily transporter ABCH in insecticidal applications.

[0024] The insecticide is to kill the larvae of red flour beetle or the larvae of diamondback moth.

[0025] The present invention uses different detergents to purify proteins, detects the ATP hydrolysis activity of ABCH in different detergent environments, and finds that in the LMNG (chemical name: 2-[6-[2-Decyl-2-[[3,4-dihydroxy-6-(hydroxymethyl)-5-[3,4,5-trihydroxy-6-(hydroxy methyl)oxan-2-yl]oxyoxan-2-yl]oxymethyl]dodecoxy]-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol) purification environment, the ATP hydrolysis activity of ABCH is reduced. Therefore, the LMNG molecule can be used as a potential insecticide targeting ABCH ( Figure 1 ).

[0026] The three-dimensional structure of the insect ATP-binding cassette superfamily transporter ABCH and LMNG complex was analyzed by cryo-electron microscopy. The three-dimensional 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 ( Figure 2 ); the ATP binding and hydrolysis domains are connected to the transmembrane domain by a connecting helix (CnH). Two LMNG binding sites are formed at the interface between the ATP binding and hydrolysis domains and the transmembrane domains of the two monomers. The binding sites include the amino acid residues on the ATP binding loop, connecting helix (CnH), transmembrane helix 1 (TM1), transmembrane helix 2 (TM2), transmembrane helix 3 (TM3) and transmembrane helix 5 (TM5) ( Figure 3 ).

[0027] The substrate binding sites are: E155, K197 amino acid residues from the ATP binding loop, N401, R404 amino acid residues from the connecting helix, F414 amino acid residue from the transmembrane helix TM1, A612, L613 amino acid residues from the transmembrane helix TM2, H644, Q648 amino acid residues from the transmembrane helix TM3, and L702, R704, N705, Q708 amino acid residues from the transmembrane helix TM5 ( Figure 3 ).

[0028] The present invention also evaluates the inhibitory ability of LMNG on ABCH by measuring IC 50 , indicating that the half-maximal inhibitory concentration of LMNG on ABCH is 58.6μΜ. ( Figure 4 ).

[0029] The present invention evaluates the insecticidal activity of LMNG against larvae of red flour beetle and larvae of diamondback moth. The results show that 500ppm LMNG can cause a 60% mortality rate ( Figure 5 ) 1000ppm LMNG can cause 90% mortality to diamondback moth larvae

[0030] The present invention takes the red castanet adenosine triphosphate-binding cassette superfamily transporter ABCH 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 to obtain high-purity protein. By applying different detergents to purify the protein, protein samples under different detergent environments are obtained, and cryo-electron microscopy samples under LMNG environment are prepared. The complete structure of the ABCH and LMNG complex is analyzed by cryo-electron microscopy technology.

[0031] The present invention is based on the determination of ATP hydrolysis activity, and it is found that LMNG may be an inhibitor of ABCH. The structure of the ABCH and LMNG complex is further analyzed, and it is found that two LMNG molecules are bound to the interface of the adenosine triphosphate binding and hydrolysis domains and the transmembrane domains of the two monomers. At the same time, through the inhibition activity detection experiment, it is shown that LMNG has significant inhibitory activity on ABCH. Through the insecticide experiment, it is shown that 500ppm LMNG can cause significant lethality to red castaneum, and 1000ppm LMNG can cause significant lethality to diamondback moth larvae. The LMNG molecule interacts with the amino acid residues on the ATP binding loop, connecting helix, transmembrane helix 1, transmembrane helix 2, transmembrane helix 3 and transmembrane helix 5 through extensive interactions, locking the ABCH protein, preventing the binding and hydrolysis of ATP, thereby inhibiting the transport function of ABCH. Therefore, this binding site can be used as a target for insecticide design to guide the design and development of efficient inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the ATP hydrolysis activity of ABCH protein in different detergent environments;

[0033] Figure 2 The overall structure of the ABCH and LMNG complex is shown as a ribbon model, the LMNG molecule is shown as a ball-and-stick model, and the box indicates the approximate location of the cell membrane;

[0034] Figure 3 is the amino acid residue of the LMNG binding site;

[0035] Figure 4 is the half-maximal inhibitory concentration curve of LMNG on ABCH;

[0036] Figure 5The insecticidal activity of LMNG against larvae of Tribolium castaneum and Plutella xylostella. DETAILED DESCRIPTION

[0037] 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.

[0038] The reagents used in the examples are all commercially available.

[0039] The amino acid sequence of the adenosine triphosphate binding cassette superfamily transporter ABCH selected from the red castaneum insect is shown in SEQ ID No.1.

[0040] Example 1: Preparation method of insect ATP-binding cassette superfamily transporter ABCH

[0041] 1. Construction of ABCH expression vector

[0042] (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.

[0043] (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.

[0044] (3) The recombinant plasmid was transformed into Escherichia coli DH5α competent cells using the chemical transformation method to obtain a cloning strain.

[0045] 2. Expression of ABCH protein

[0046] (1) Plasmid extraction

[0047] 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.

[0048] (2) Recovery and passaging of HEK293F cells

[0049] 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 .

[0050] (3) HEK293F cell transfection

[0051] HEK293F cells were transfected using a linear PEI cationic polymer transfection method. The transfection method is as follows:

[0052] 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 cells, 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, and let it stand for 30min to allow the PEI-DNA complex to form. 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, and then culture it at 150rpm, 37℃, 5% carbon dioxide for 2 days, and collect the cells by centrifugation at 1000g for 5min.

[0053] 3. Purification of ABCH Protein

[0054] (1) The collected HEK293F cells were suspended in 20mM Tris-HCl (pH 8.0), 150mM NaCl buffer, and the cells were broken with a high-pressure homogenizer, and then centrifuged at 13000rpm for 30min, and the supernatant was collected. Ultracentrifugation was performed at 150000×g to collect the cell membrane. The cell membrane was dissolved in 20mM Tris-HCl (pH 8.0), 150mM NaCl, and 1% different detergents for 1h (LMNG was used to dissolve the cell membrane when preparing ABCH and LMNG complex cryo-EM samples) to fully dissolve the target protein on the cell membrane, and the mixed components were ultracentrifuged at 150000×g to collect the supernatant.

[0055] (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.

[0056] (3) After the protein is collected, it is concentrated to 500 μL and further purified using a Superose 6 gel filtration column to obtain a high-purity target protein. Through the purification steps above, a high-purity ABCH protein is obtained, which can be used for the preparation of cryo-electron microscopy samples.

[0057] Example 2: Effects of different detergent environments on the activity of insect ATP-binding cassette superfamily transporter ABCH

[0058] The ATPase activity of purified ABCH in different detergents was measured at 30°C. The reaction system contained 50mMTris (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. The results showed that the activity of ABCH was significantly reduced under the LMNG environment ( Figure 1 ).

[0059] Example 3: Detection of the half-maximal inhibitory concentration of LMNG on insect ATP-binding cassette superfamily transporter ABCH

[0060] ABCH protein was incubated with different LMNG concentrations for 10 minutes, and then 5mM PEP, 20U / ml pyruvate kinase, and 2mM ATP were added to the system. The reaction buffer contained 50mM Tris (pH 8.0), 2mM MgCl2, 150mM KCl, 1mM DTT, and 0.03% Glyco-diosgenin. After 30 minutes, the reaction was stopped by adding 100μL of a solution containing 0.36mM 2,4-dinitrophenylhydrazine and 0.4MHCl, and incubated at 37°C for 15 minutes. Finally, 100μL of 2.5MNaOH 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. The inhibition curve was drawn with the Log value of the LMNG concentration as the horizontal axis and the ATPase activity corresponding to different concentrations of LMNG as the vertical axis. The results showed that the IC of LMNG for ABCH 50 The value is 58.6 μM ( Figure 4 ).

[0061] Example 4: Preparation of frozen samples of the complex of Tribolium castaneum ABCH and LMNG

[0062] (1) During the purification process, the cell membrane was dissolved with LMNG and then incubated in 50 mM Tris (pH 8.0), 150 mM NaCl, 2 mM DTT, 2 mM Mg 2+ , gel filtration was performed under 0.05% Glyco-diosgenin conditions to obtain high-purity ABCH protein, which was concentrated to 5 mg / mL;

[0063] (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.

[0064] (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).

[0065] (4) Take 3.5 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.

[0066] Example 5: Structural analysis of the complex of Tribolium castaneum ABCH and LMNG

[0067] 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.

[0068] For the ABCH and LMNG complex sample, a total of 5210 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 measured at 100 μm resolution, and then the structure was constructed and fine-tuned using PHENIX and Coot according to the protein sequence, and the structure was evaluated using the PDB Validation Service (see Table 1 for structural parameters).

[0069] Table 1 Cryo-EM data collection and structure correction data statistics

[0070]

[0071] The three-dimensional structure of the insect ATP-binding cassette superfamily transporter ABCH and LMNG complex was analyzed by cryo-electron microscopy. The three-dimensional 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 ( Figure 2 ); the ATP binding and hydrolysis domains are connected to the transmembrane domain by a connecting helix (CnH). Two LMNG binding sites are formed at the interface between the ATP binding and hydrolysis domains and the transmembrane domains of the two monomers. The binding sites include the amino acid residues on the ATP binding loop, connecting helix (CnH), transmembrane helix 1 (TM1), transmembrane helix 2 (TM2), transmembrane helix 3 (TM3) and transmembrane helix 5 (TM5) ( Figure 3 ).

[0072] The binding site substrate binding site is: E155, K197 amino acid residues from the ATP binding loop, N401, R404 amino acid residues from the connecting helix, F414 amino acid residue from the transmembrane helix TM1, A612, L613 amino acid residues from the transmembrane helix TM2, H644, Q648 amino acid residues from the transmembrane helix TM3, and L702, R704, N705, Q708 amino acid residues from the transmembrane helix TM5 ( Figure 3 ).

[0073] Example 6: Insecticidal activity of LMNG against Tribolium castaneum and Plutella xylostella larvae

[0074] The larvae of Red Castanea were placed on a feed containing 500ppm LMNG, and the mortality of the larvae was measured after 7 days. The preparation method of the Red Castanea feed is as follows: 30 ml of an aqueous solution containing 500ppm LMNG is mixed with 30 grams of wheat flour. After the mixture is stirred for 30 minutes, it is freeze-dried for five days and then ground into powder. The control feed is prepared in the same way, but no small molecules are added. The larvae of Plutella xylostella were placed on a feed containing 1000ppm LMNG, and the mortality of the larvae was measured after 7 days. The Plutella xylostella feed uses commercial artificial feed. The results show that LMNG has obvious insecticidal activity against Red Castanea and Plutella xylostella larvae ( Figure 5 ).

Claims

1. The structure of the insect ATP-binding cassette superfamily transporter ABCH and LMNG complex is 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. Two LMNG binding sites are formed at the interface between the ATP binding and hydrolysis domains and the transmembrane domains of the two monomers, which contain amino acid residues on the ATP binding loop, the connecting helix, transmembrane helix 1, transmembrane helix 2, transmembrane helix 3, and transmembrane helix 5.

2. The insect ATP-binding cassette superfamily transporter ABCH and LMNG complex structure according to claim 1, wherein the insect is Tribolium castaneumHerbst; 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 and LMNG complex structure described in claim 2, the binding site substrate binding site is: E155, K197 amino acid residues from the ATP binding loop, N401, R404 amino acid residues from the connecting helix, F414 amino acid residue from the transmembrane helix TM1, A612, L613 amino acid residues from the transmembrane helix TM2, H644, Q648 amino acid residues from the transmembrane helix TM3, and L702, R704, N705, Q708 amino acid residues from the transmembrane helix TM5.

4. Application of insect adenosine triphosphate-binding cassette superfamily transporter ABCH as insecticide target.

5. The application according to claim 4, comprising the following steps: (a) Based on the ATP hydrolysis experiment, the effects of different detergents on the ATP hydrolysis activity of insect ATP-binding cassette superfamily transporter ABCH were screened, and detergents that inhibit the activity of insect ATP-binding cassette superfamily transporter ABCH were obtained. (b) Preparation of composite cryo-EM samples (I) The high-purity ABCH protein obtained in a detergent environment that inhibits the activity of insect ATP-binding cassette superfamily transporter ABCH was concentrated to 5 mg / mL; (II) The R1.2 / 1.3, 300-mesh gold grid produced by Quantifoil was glow-discharged for 30 seconds, and the glow-discharge gas was H2 and O2; (III) The sample was prepared using FEI's Vitrobot Mark IV, and the freezing sample preparation conditions were set as follows: blot-time: 3s, wait-time: 5s, drain-time: 0s, blot-force: 3; (IV) 3.5 μL of protein was taken and added to the surface of the grid. After the Vitrobot Mark IV completed the sample preparation procedure, the sample was quickly placed in liquid ethane and then stored in liquid nitrogen; (c) The frozen samples were collected and analyzed under a cryo-electron microscope to obtain the three-dimensional structure of the protein ABCH and the detergent binding site that inhibits the activity of the insect ATP-binding cassette superfamily transporter ABCH; (d) The detergent binding site that inhibits the activity of insect adenosine triphosphate-binding cassette superfamily transporter ABCH in the three-dimensional structure is used as a target to guide the design and development of insecticides targeting ABCH.

6. The use according to claim 5, wherein the detergent molecule is LMNG, and the binding sites are E155, K197 amino acid residues from the ATP binding loop, N401, R404 amino acid residues from the connecting helix, F414 amino acid residue from the transmembrane helix TM1, A612, L613 amino acid residues from the transmembrane helix TM2, H644, Q648 amino acid residues from the transmembrane helix TM3, and L702, R704, N705, Q708 amino acid residues from the transmembrane helix TM5.

7. The use according to claim 5, wherein the method for preparing the high-purity ABCH protein in step (I) is: using HEK293F cells to recombinantly express the ABCH protein, disrupting the collected ABCH-expressing cells, and obtaining the high-purity ABCH protein using affinity chromatography and gel filtration chromatography.

8. The use according to claim 7, wherein the affinity chromatography and gel filtration chromatography comprise 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% LMNG 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.

9. The use according to claim 7, wherein the recombinant expression of ABCH protein in step (I) 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, 150 rpm, and 5% CO2.

10. Use of LMNG as an inhibitor of insect adenosine triphosphate-binding cassette superfamily transporter ABCH in killing insects, wherein the insecticide is killing larvae of red flour beetle or diamondback moth.

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