ATG8 as drug target for screening and preventing fruit fly pests and application of ATG8

Abstract

The invention discloses ATG8 protein as a target for screening drugs for preventing and treating fruit fly pests and application of the ATG8 protein. The invention provides a target for screening drugs for preventing and treating fruit fly pests. The target is autophagy related protein 8, namely ATG8; the invention also provides an application of the autophagy-associated protein 8, namely ATG8, as a drug target in screening drugs for preventing and treating fruit fly pests. The invention further provides an insecticidal drug taking the ATG8 as the target. Aiming at the importance of ATG8 on the vital activities of insects, ATG8 is used as an action target of a potential insecticide for the first time, drug active small molecules are subjected to in-vitro screening, an ATG8a crystal structure of the drosophila melanogaster is analyzed, the insecticidal activity of the screened compound on the drosophila melanogaster is determined, and the ATG8 is proved to be a potential insecticide target indeed.

Description

Technical Field

[0001] The invention relates to ATG8 as a drug target for screening and controlling fruit fly pests and application thereof, belonging to the technical field of pest control. Background Art

[0002] Drosophila melanogaster is an important borer pest of blueberry fruits during the color change and ripening period. It is fond of feeding on ripe and rotten blueberry fruits. The higher the maturity, the more serious the damage. When blueberry fruits are damaged, they are not only very easy to fall off and rot, greatly shortening the shelf life of the fruit, but also causing consumers to have a greater loss of appetite, seriously affecting the benefits of blueberry cultivation.

[0003] Drosophila melanogaster not only causes great harm to fruits, but also has a huge impact on edible fungi. Drosophila melanogaster can harm a variety of edible fungi such as hairy fungus, white fungus, black fungus, mushrooms, Agrocybe oleraceus, and Pleurotus ostreatus. The larvae feed on mycelium and culture medium, causing the surface of the fungus bag to become water-stained and rotten, and bore into the fruiting body, causing the mushroom stem and mushroom cap to rot. In severe cases, the fungus harvest may be completely lost.

[0004] At present, the method of controlling Drosophila melanogaster is still mainly chemical control: insecticides. However, due to the extensive use of insecticides, many Drosophila melanogaster have developed serious resistance to multiple insecticides, resulting in a significant reduction in the effectiveness of insecticides. In this case, people often increase the amount of insecticides used to control Drosophila melanogaster, which seriously pollutes the environment.

[0005] Therefore, it is of great significance to use Drosophila melanogaster as experimental material and then develop new insecticide targets to better control fruit fly pests. Summary of the invention

[0006] Based on the above, the present invention provides ATG8 as a drug target for screening and controlling fruit fly pests and its application.

[0007] The technical solution of the present invention is:

[0008] In a first aspect, the present invention provides a target for screening drugs for controlling fruit fly pests, wherein the target is autophagy-related protein 8, namely ATG8.

[0009] In a second aspect, the present invention provides an application of autophagy-related protein 8, namely ATG8, as a drug target in screening drugs for controlling fruit fly pests.

[0010] In a third aspect, the present invention provides a drug for screening and controlling fruit fly pests by using autophagy-related protein 8, namely ATG8, as a target for screening, wherein the drug is 6F indole.

[0011] Technical principle of the present invention: ATG8 is essential for the normal physiological life of fruit flies. Autophagy is a process in which cells autonomously degrade through lysosomes to achieve the recycling of intracellular substances, and plays an important role in the cell differentiation and individual development of fruit flies. If there is a strong interaction between highly active small molecules and target proteins, it will directly lead to the inability of proteins to exert their corresponding physiological activities, which will directly affect the autophagy process of fruit flies, and then destroy the normal growth and development of fruit flies, which will greatly reduce the harm of fruit flies to plants.

[0012] Beneficial effects of the present invention: In view of the importance of ATG8 protein to the life activities of fruit flies, the present invention uses ATG8 as a potential target of insecticides for the first time, screens drug-active small molecules in vitro, and searches for active binding sites between the drug and ATG8 after molecular docking the screened drug small molecules with ATG8. At the same time, the insecticidal activity of the active small molecules on Drosophila melanogaster is determined by using a combined toxicity assay for stomach poison contact killing, proving that ATG8 can indeed become a potential target of insecticides. In addition, the drug 6F indole screened by the present invention has a good control effect on fruit flies. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Gel electrophoresis of DMATG8a protein;

[0014] Figure 2 The three-dimensional structure of DMATG8a;

[0015] Figure 3 Two active pockets of DMATG8a;

[0016] Figure 4 MST was used to measure the binding affinity of DMATG8a to 6F-indole;

[0017] Figure 5 Molecular docking results of DMATG8a and 6F-indole. DETAILED DESCRIPTION

[0018] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0019] 1. Experimental Materials

[0020] (I) Plasmids and expression strains

[0021] The pET32a-DmATG8a plasmid was constructed by Beijing Qingke Biotechnology Co., Ltd.

[0022] The BL21(DE3) strain was purchased from Shanghai Shenggong Biological Technology Co., Ltd.

[0023] Drosophila melanogaster was purchased from Guangzhou Excellence Angel Biological Laboratory.

[0024] (II) Test proteins and insecticidal small molecules

[0025] DmATG8a protein was expressed and purified; halogenated indole compounds were purchased from Shanghai Aladdin Reagent Company.

[0026] (III) Buffer solution and its preparation method

[0027] LB solid medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 8 g agar powder, 1000 mL secondary water, shake thoroughly to mix evenly, adjust the pH to 7.4, sterilize at 121°C for 20 min, and cool to room temperature for use.

[0028] LB liquid culture medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1000 mL secondary water, shake thoroughly to mix evenly, adjust the pH to 7.4, sterilize at 121°C for 20 min, and cool to room temperature for use.

[0029] 1M Tris-HCl solution: Weigh 121.14 g of Tris solid, add 800 mL of secondary water, adjust the pH to 8.0 with hydrochloric acid after complete dissolution, and finally dilute to 1000 mL with secondary water.

[0030] 75% ethanol solution: Measure 750mL of anhydrous ethanol solution, add 250mL of secondary water, and stir well.

[0031] 0.1M CaCl2 solution: Weigh 11.1g CaCl2 solid, add 1000mL secondary water, and stir to dissolve.

[0032] 2× polyacrylamide gel loading buffer: Weigh 0.4 g of SDS solid, 0.1 mg of bromophenol blue solid, 2.5 mL of 0.5 M Tris-HCl (PH 6.8), 2 mL of glycerol, and 0.2 mL of β-mercaptoethanol, and finally add 5.2 mL of secondary water, shake thoroughly to mix well, and place at room temperature for use.

[0033] 5× electrophoresis buffer: Weigh 15.1 g of Tris solid, 94 g of glycine, and 5.0 g of SDS respectively, add about 800 mL of secondary water, shake thoroughly to mix evenly, and finally make up to 1000 mL and store at room temperature for later use.

[0034] Binding buffer solution (bufferA): 50 mM Tris, 150 mM NaCl, 20 mM Imidazole, pH 8.0.

[0035] Elution buffer solution (buffer B): 50 mM Tris-HCl, 150 mM NaCl, 300 mM Imidazole, pH 8.0.

[0036] Lysis buffer: 50 mM Tris-HCl, 150 mM NaCl, pH 8.0.

[0037] Desalting buffer: 50 mM Tris-HCl, 150 mM NaCl, pH 7.5.

[0038] 2. Construction of DmATG8a vector

[0039] The pET32a-DmATG8a plasmid was constructed by Beijing Qingke Biotechnology Co., Ltd.

[0040] 3. Expression and purification of DmATG8a

[0041] (I) Preparation of competent cells

[0042] 1. Pick a single clone from the newly activated plate and inoculate it into a clean centrifuge tube containing 100 mL of LB medium. Incubate it on a shaker at 37°C and 200 rpm for about 4 hours until the OD600 reaches 0.45.

[0043] 2. Place the sample on ice for about 10 minutes;

[0044] 3. Centrifuge the bacterial solution for 15 minutes at 4°C and 4000 rpm.

[0045] 4. Discard the supernatant and add 30 mL of pre-cooled 0.1 M CaCl2 solution to fully resuspend the cells;

[0046] 5. Centrifuge the mixture for 4 min at 4°C and 5000 rpm.

[0047] 6. Discard the supernatant and add 2 mL of 0.02 M CaCl2-0.08 M MgCl2 solution pre-cooled at 4°C to resuspend the cells;

[0048] 7. Aliquot the resuspended cell suspension, freeze it quickly with liquid nitrogen, and store it in a -80℃ refrigerator for later use.

[0049] (II) Transformation of pET32a-DmATG8a recombinant plasmid

[0050] 1. Under sterile conditions in a clean bench, take 100 μL of competent cells BL 21 (DE 3) and add 2 μL of pET32a-DmATG8a plasmid;

[0051] 2. Place the sample on ice for about 30 minutes;

[0052] 3. Set the water bath to 42°C and heat shock the sample for 80 seconds after the temperature reaches 42°C;

[0053] 4. Place the sample on ice for about 2 minutes;

[0054] 5. Add 900 μL of LB liquid medium containing ampicillin sodium to the centrifuge tube containing the sample;

[0055] 6. Place the bacterial solution in a shaker and culture for 45 minutes at 150 rpm;

[0056] 7. Centrifuge the bacterial solution in a centrifuge for 5 minutes at 4000 rpm. After centrifugation, discard 900 μL of the supernatant, fully resuspend the bacteria, and use a pipette to draw 100 μL of the bacterial solution and apply it on the LB solid plate;

[0057] 8. Place the culture dish in a 37°C incubator for overnight culture.

[0058] (III) Expression of DmATG8a

[0059] 1. Pick a single clone from the plate with a single clone colony, add 10 mL of LB liquid medium and 30 ng / mL kanamycin to a sterilized 50 mL centrifuge tube, and culture overnight at 37°C and 200 rpm on a shaker;

[0060] 2. When the OD600 of the test bacterial solution reaches 0.65, add 1M IPTG and then culture on a shaker at 16°C and 200 rpm for about 14 hours;

[0061] 3. Centrifuge the bacterial solution in a centrifuge for 25 minutes at 5000 rpm, discard the supernatant, and collect the bacterial cells;

[0062] 4. Place on ice for about 10 minutes, add about 40 mL of lysis buffer solution to the centrifuge tube, and resuspend on ice for 10 minutes;

[0063] 5. Use an ultrasonic cell disruptor to disrupt the cells. The ultrasonic conditions are set as follows: 40% power, 4 seconds on, 6 seconds off, and continuous ultrasonication for 35 minutes;

[0064] 6. Place the sonicated suspension into a sterilized centrifuge tube and centrifuge at 4°C, 12,000 rpm for 30 min. Keep the supernatant and perform 12% SDS-PAGE gel electrophoresis on 20 μL of the supernatant.

[0065] (IV) Purification of DmATG8a

[0066] 1. Filter the prepared solution (binding buffer and elution buffer) with a 0.22 μm water-based microporous filter membrane to remove solid impurities and remove bubbles by ultrasonication;

[0067] 2. Clean the peristaltic pump with 20% ethanol solution for about 10 mL, and then clean the pump with secondary water for about 10 mL. After cleaning, rinse the Ni column with Buffer A for about 20 mL, and pass the supernatant obtained by centrifugation through the Ni column for about 2 hours;

[0068] 3. Use a protein affinity chromatography system to perform gradient elution on the Ni column;

[0069] 4. After elution, clean the purification system with 20% ethanol solution and secondary water, about 50 mL;

[0070] 5. Perform 12% SDS-PAGE gel electrophoresis on the protein effluent at the peak position to screen the effluent containing the target protein;

[0071] 6. Use a 10KDa ultrafiltration tube to concentrate the target protein;

[0072] 7. Use a desalting column to desalt the concentrated target protein:

[0073] 8. The obtained protein was again subjected to 12% SDS-PAGE gel electrophoresis detection, and the target protein was quickly frozen with liquid nitrogen and stored in a -80°C refrigerator.

[0074] 4. Molecular weight verification of DmATG8a

[0075] The protein effluent at the peak position during purification and the sample after desalting were boiled and denatured, and then electrophoresed using 15% SDS-PAGE. After purification by Ni-NTA affinity column, a clear band was obtained, the size of which was consistent with the size of the target protein 18KDa ( Figure 1 ).

[0076] The electrophoresis steps are as follows:

[0077] 1. Add 20 mL of protein sample to 20 mL of 2×SDS loading buffer and mix thoroughly;

[0078] 2. Place the mixture in a water bath at 100°C for 10 minutes;

[0079] 3. Add 15 μL of denatured sample to the sample tank of the gel and set the electrophoresis conditions to: 90V for 20 min for the concentrated gel and 120V for about 80 min for the separated gel;

[0080] 4. After electrophoresis, take out the gel and stain it with Coomassie Brilliant Blue dye. After staining, decolorize it with Coomassie Brilliant Blue decolorizing solution. After decolorization, observe the protein bands.

[0081] Table 1 Preparation of 15% SDS-PAGE electrophoresis gel

[0082]

[0083] 5. Crystallization of DmATG8a

[0084] 1. Use ultrafiltration tube to concentrate the desalted DMATG8a to 18 mg / mL;

[0085] 2. Crystallization conditions were searched using the sessile drop vapor diffusion method at 18 °C using Crystal Screens I and II, Index I and II (Hampton Research);

[0086] 3. After 2 days, use a microscope to observe whether there are crystals in the pool liquid;

[0087] 4. Record the pool solution of the growing crystals, and adjust the salt concentration, precipitant concentration and pH to optimize the crystals;

[0088] 5. Select high-quality crystals to collect data at the Shanghai Synchrotron Radiation Source;

[0089] 6. Data were processed using Phenix, CCP4 and Coot software.

[0090] After experiments, the crystal structure of DMATG8a was refined into Resolution. DMATG8a crystals are composed of 121 amino acids and 130 water molecules, with a total of 4 β-folds (β1-β4) and 5 α-helices (α1-α5), of which the 4 β-folds are in the middle, and the 5 α-helices are divided into α1, α2 and α3, α4, α5, distributed on both sides of the β-fold. The overall structure of the protein can be divided into two parts, one is the N-terminal α-helical structure (α1, α2), and the other is the core area of ​​the ubiquitination fold, which is composed of 4 β-folds and 3 α-helices (α3, α4 and α5) ( Figure 2 ).

[0091] Two pockets were found in DMATG8a. Pocket 1 is located between α-helix 2 and the ubiquitination folding region and is composed of Glu17, Ile21, Pro30, Lys48, Leu50 and Phe104. Pocket 2 is located within the ubiquitination folding region and is composed of Tyr49, Val51, Pro52, Phe60, Leu63 and Ile64. Figure 3 ).

[0092] VI. Study on the interaction between DmATG8a and halogenated indole compounds

[0093] MST studied the interaction between DmATG8a protein and halogenated indole compounds. The interaction between halogenated indole and DmATG8a was studied using Monolith NT.115 microthermophoresis.

[0094] Before the experiment, the drug was diluted in a concentration gradient to prepare 16 different concentrations of drug solution (500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, 0.24, 0.12, 0.06, 0.03, 0.015 μM). The prepared drug was incubated with fluorescent dye-labeled DmATG8a in the dark for about 30 min.

[0095] The instrument parameters of MST should be set to: LED power 40%, Laser power 30%, Red excitation.

[0096] MST studies have shown that 6F indole has a strong interaction with DMATG8a. The dissociation constant between 6F indole and DMATG8a is Kd = 3.54 μM ( Figure 4 ).

[0097] The DMATG8a structure obtained above was molecularly docked with the active small molecule. The molecular docking results are shown in Figure 56F-indole forms a hydrogen bond with Lys48 of DMATG8a, which is a residue in the tryptophan binding cavity of DMATG8a, so 6F-indole may have good binding activity with pocket 1 of DMATG8a.

[0098] VII. Gastric poison and contact toxicity assay to determine the insecticidal activity of active small molecules against Drosophila melanogaster

[0099] 1. Dissolve the active small molecule in acetone to prepare a mother solution with a concentration of 10 mg / mL;

[0100] 2. Dilute it with sterile water containing 0.5% Tween 80 to different concentration gradients of 500 μg / mL, 250 μg / mL, 125 μg / mL, 62 μg / mL, 31 μg / mL, 15 μg / mL and 7 μg / mL;

[0101] 3. Lay a piece of filter paper on the bottom of a 9cm petri dish, and take 0.8ml of the prepared test solution of the corresponding concentration and drip it on the surface of the filter paper to completely wet it. Soak the cut apple slices of about 2cm*2cm*3mm in the test solution of different concentrations for 1min, then take them out and place them in a petri dish covered with filter paper treated with the same solution;

[0102] A 4.0-number brush was used to gently pick up two-day-old adults of Drosophila melanogaster of the same size. 20 adults were treated with each concentration, and each treatment was repeated 3 times, with clean water as the control. After treatment, the insects were placed in an incubator with a temperature of (24±0.1)℃, a humidity of 75%±5%, and a light cycle of L:D=16:8. The death of the insects was checked and recorded 24 hours after the drug was applied. The insect body was lightly touched with a brush, and the inability to move was recorded as death.

[0103] The results showed that 6F indole showed 100% insecticidal activity against Drosophila melanogaster at a concentration of 500 μg / mL. However, as the concentration decreased, the insecticidal activity of 6F indole against Drosophila melanogaster showed a large change trend. When the concentration was 125 μg / mL, only 21.67% of Drosophila melanogaster died, while at a concentration of 31 μg / mL, it completely lost its activity against Drosophila melanogaster. The final half maximum lethal concentration (LC50) of 6F indole was 1.34. 50 ) was 166.6μg / mL.

[0104] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A target for screening drugs for controlling fruit fly pests, characterized in that: The target is autophagy-related protein 8, namely ATG8.

2. Application of autophagy-related protein 8, i.e. ATG8, as a drug target in screening drugs for controlling fruit fly pests.

3. A drug for controlling fruit fly pests screened using autophagy-related protein 8, i.e. ATG8, as a target, wherein the drug is 6F indole.

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