Peptide compound for preventing and treating aphids as well as preparation method and application of peptide compound

Abstract

The invention discloses a peptide compound for preventing and treating aphids as well as a preparation method and application thereof, and relates to the technical field of biology. The structural formula of the peptide compound is # imgabs0 #. The insect neuropeptide Kinin is subjected to structural modification, computer-aided design is combined, the titer of the modified compound is simulated and evaluated, and a No.2 amino acid side chain group of the Kinin neuropeptide is modified, so that the peptide compound is synthesized. The peptide compound has binding activity with a Kinin receptor and aphid killing activity, can influence functions of neuropeptide receptors of pests and further influence growth and development of the neuropeptide receptors so as to realize control of pest populations, and has lower non-target effect, higher accuracy and environmental friendliness and wide application prospects compared with traditional pesticides.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a peptide compound for preventing and controlling aphids, and a preparation method and application thereof. Background Art

[0002] Aphids belong to the order Hemiptera, including two major categories: Adelgoide and Aphidoidea, with 13 families and more than 500 genera. At present, there are nearly 5,000 known species of aphids in the world, and more than 1,000 species have been found in my country. Aphids are one of the main pests of plants, which are very harmful to plants and cause huge economic losses to agricultural production. On the one hand, aphids directly harm plants by sucking plant sap or releasing toxic substances, affecting the normal physiological metabolic activities and growth and development of plants. On the other hand, aphids are the transmission medium of plant pathogens. Most of the extremely common viral diseases of plants are transmitted by aphids, which greatly increases the harm of aphids to plants. In addition, aphids have strong reproductive ability, can reproduce sexually and parthenogenetically, have a short generation cycle, a large number, and a very serious phenomenon of generation replacement. Therefore, aphid control is very difficult.

[0003] At present, the prevention and control methods of aphids are still mainly chemical, with agricultural and physical control. How to carry out comprehensive prevention and control of aphids in a simple, green, safe, low-cost and high-efficiency manner is a problem that still needs to be solved in plant protection technology. Biological control, as a relatively new prevention and control measure, is gradually being developed and used by people. As early as the last century, there were many records of the use of biological pesticides. Biological pesticides are mainly derived from microorganisms and their product preparations, plant products and their extracts, natural enemies and some genetically modified substances. At present, biological pesticides are widely used worldwide and have been widely used in agricultural production. However, the application history in my country is relatively short, and relevant knowledge and technology are lacking. The development speed was relatively slow before, so it is of great significance to develop new green biological pesticides.

[0004] Neuropeptides, as a natural biochemical active substance, are characterized by high efficiency in trace amounts and strong specificity, and play an important role in regulating physiological functions in organisms. The active substance has strong targeting, and the raw material is a polypeptide synthesized from natural amino acids, which is a suitable target for the development of green biological pesticides. Research results have shown that the insect neuropeptide Kinin has the function of regulating insect feeding and diuresis. On this basis, the present invention intends to structurally modify the insect neuropeptide Kinin to develop a new neuropeptide compound with aphid-killing activity. Summary of the invention

[0005] The purpose of the present invention is to provide a peptide compound for controlling aphids and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The peptide compound has binding activity with Kinin receptors and aphid-killing activity, which can affect the function of neuropeptide receptors of pests, thereby affecting their growth and development, thereby achieving control of pest populations. Compared with traditional pesticides, it has lower non-target effects and higher precision and environmental friendliness, and has broad application prospects.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a peptide compound for controlling aphids, the structural formula of which is as follows:

[0008]

[0009] The present invention also provides a method for preparing the above-mentioned peptide compound, comprising the step of synthesizing the peptide compound by solid phase synthesis.

[0010] The present invention also provides the use of the above peptide compounds in the preparation of insecticides.

[0011] Furthermore, the pests and diseases targeted by the pesticide are aphids.

[0012] Furthermore, the aphid is Myzus persicae.

[0013] The present invention also provides an insecticide, the active ingredient of which includes the above-mentioned peptide compound.

[0014] Furthermore, the pesticide also includes a pesticide adjuvant.

[0015] Furthermore, the pesticide adjuvant includes a dispersant, a cosolvent, a stabilizer, a wetting agent or a spreader.

[0016] The present invention also provides a method for preventing and controlling aphids, comprising the step of spraying the above-mentioned peptide compound or insecticide on plant strains.

[0017] The effective spraying dose of the peptide compound is ≥0.1 μM.

[0018] The present invention discloses the following technical effects:

[0019] The present invention structurally modified the insect neuropeptide Kinin, combined with computer-aided design, simulated and evaluated the potency of the modified compound, modified the side chain group of amino acid No. 2 of the Kinin neuropeptide, and synthesized a peptide compound, L02. Subsequently, an in vitro cell calcium ion flow experiment was conducted on it, confirming that the compound has binding activity with the Kinin receptor, and a direct contact toxicity assay experiment with aphids confirmed that it has aphid-killing activity.

[0020] This invention synthesized for the first time a neuropeptide analogue with aphidicidal activity - L02, and pioneered a new method for controlling aphids, a harmful agricultural organism. It also used computer-aided design technology to develop a new type of biological insecticide with natural amino acids as raw materials. Compared with traditional chemical pesticides, it has higher precision and environmental friendliness.

[0021] The present invention utilizes the principle of high target specificity of neuropeptides to develop antagonist neuropeptide analogs, which can affect the function of neuropeptide receptors of pests and further affect their growth and development, thereby achieving control of pest populations. Compared with traditional pesticides, it has lower non-target effects, higher precision and environmental friendliness, and has broad application prospects.

[0022] The raw material of the invention is amino acid, which can reduce the pollution and influence of chemical pesticides on the environment and non-target organisms, and is helpful to protect the health of the ecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 is the structural formula diagram of compound L02;

[0025] Figure 2 This is a statistical graph of the results of the cell calcium ion flow experiment;

[0026] Figure 3 This is a statistical chart of the direct contact toxicity test results of compound L02 on green peach aphid; where **** represents P≤0.0001. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1. Modification of natural insect neuropeptide Kinin

[0034] Computer molecular docking analysis of natural insect neuropeptide Kinin and its receptor revealed that the key binding site, serine II, did not have a strong binding force, so this site was modified. A new compound L02 was obtained by introducing a long-chain hydroxyl group. The structural formula is shown in Figure 1 .

[0035] 2. Activity prediction of compounds

[0036] The SYBYL-X2.0 software was used to construct a 3D-QSAR model for the activity database, and the structures and activity data of 44 compounds in the database were imported. The database compounds were superimposed on the model molecular skeleton model, and then the CoMFA model was calculated. The obtained CoMFA model values ​​were subjected to PLS analysis to obtain a good prediction model.

[0037] The new compound L02 designed by the present invention was imported into the software SYBYL-X2.0 for model molecular skeleton model superposition. The prediction model was used to perform 3D-QSAR activity prediction on the unknown active compound L02 designed by the present invention, and it was found that L02 had a higher activity value.

[0038] Example 2

[0039] Synthesis of modified compound L02:

[0040] Peptide synthesis uses SPPS solid phase synthesis method. Using Fmoc chemistry, amino acids are added to the resin sequentially to build the peptide chain. After the synthesis is completed, the Fmoc group at the N-terminus is first deprotected, then the side chain protecting groups are deprotected, and the peptide is cleaved from the resin. The specific procedure is as follows:

[0041] 1. Coupling the first amino acid. Take an appropriate amount of modified chlorinated resin, add the pre-prepared amino acid solution into the resin and react for a period of time. Among them, the long-chain hydroxyl non-natural amino acid raw material is purchased from Anaiji Chemical Reagent Network, and its name is 2-amino-5-hydroxypentanoic acid (CAS: 6152-89-2).

[0042] 2. Add pip / DMF solution for a while and then remove the solvent by vacuum filtration.

[0043] 3. Washing after Fmoc removal: Add DMF to the resin (washing step) and then remove the solvent by vacuum filtration.

[0044] 4. QC tested the resin. The actual MW relative molecular weight was 576.4, which was consistent with the theoretical value.

[0045] Example 3

[0046] Compound L02 was used to conduct a cellular calcium ion flow experiment in the CHO WTA1 hamster ovary cell line. The experimental steps are as follows:

[0047] Cell recovery: 1 mL of the stored CHO-WTA11 cells was added to a cell culture dish (10 cm in diameter) containing 15 mL of growth medium, and then cultured in an incubator at 37°C with 5% CO2.

[0048] Cell passaging: After the cells grow to cover 80% of the culture dish, passaging is performed. First, remove the growth medium, add 32mL of trypsin, let it stand for 3 minutes, and gently pipette back and forth; add all the trypsin solution mixed with cells into a 15mL centrifuge tube, then add 76mL of growth medium, and centrifuge at 2,500900rpm for 35min; carefully remove the supernatant, add 3mL of growth medium, and mix gently; add an appropriate amount of mixed cell solution to the cell culture dish containing 15mL of growth medium, and then culture in an incubator at 37°C and 5% CO2.

[0049] Co-transfection: Add 1.5 mL of DMEM / F-12 basic culture medium to a 15 mL EP tube; add 7.5 μg of GmCrz-R-pcDNA3.1(+) plasmid and Aequorin plasmid respectively; then, add 45 μL of TransIT-LT1 transfection reagent to the EP tube, use a pipette to gently mix, and let stand at room temperature for 15 min; add the above transfection mixture dropwise to the prepared cells, and culture in an incubator at 37°C and 5% CO2 for 24 h.

[0050] Collect cells: remove the growth medium, add 3mL trypsin, let it stand for 3 minutes, and then gently pipette back and forth; add all the trypsin solution mixed with cells into a 15mL centrifuge tube and centrifuge at 2,500rpm for 3 minutes; add 3mL DMEM / F-12 basic medium to resuspend the cells; add 30uL coelenterazine (Coelenterazine) storage solution (concentration is 500μM), and stir magnetically for 3h in the dark; add 27mL DMEM / F-12 basic medium to make the final volume 30mL, stir magnetically for 30min, and use SpectraMax L chemiluminescence microplate reader to detect the chemiluminescence value. All tests include 2 technical replicates and 3 biological replicates.

[0051] Test ligand preparation: The test ligand concentration contains 8 concentration gradients, namely 10μM, 1μM, 10-1μM, 10-2μM, 10-3μM, 10-4μM, 10-5μM and 10-6μM. Enzyme-free sterile water is used as a negative control, and 0.1% TritonX100 is used as a positive control. The reaction system contains 50μL ligand and 50μL spare cells.

[0052] The results of the determination of the binding activity of compound L02 to Kinin receptor are shown in Figure 2 The results showed that compound L02 had an antagonistic effect on Kinin receptors.

[0053] Example 4

[0054] Direct contact toxicity assay against green peach aphid using L02 compound:

[0055] 1 mg of compound L02 was dissolved in 500 μL of dimethyl sulfoxide (DMSO), and 1235 μL of HPLC water was added to dilute the solution to a concentration of 1 mM. The solution was further diluted to a concentration of 1 μM with HPLC water to obtain L02 solution for biological assay.

[0056] 1 mg of neuropeptide Kinin was dissolved in 500 μL of DMSO and diluted to a concentration of 1 μM with HPLC water to obtain Kinin solution for biological assay.

[0057] Adult peach aphids were selected for toxicity determination, with 20 aphids in each group and five replicates in total. 100 nL of solution (L02 solution, Kinin solution or solvent) was dripped on each aphid and observed for absorption. The aphids were then placed in a suitable environment and the number of deaths was observed and recorded every day for 7 days to obtain aphid survival curves. Figure 3 As shown, CK is the solvent control group. The results show that compound L02 has aphidicide activity.

[0058] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A peptide compound for controlling aphids, characterized in that: The structural formula is as follows:

2. A method for preparing a peptide compound as claimed in claim 1, characterized in that: The method comprises the step of synthesizing the peptide compound by adopting a solid phase synthesis method.

3. Use of the peptide compound as claimed in claim 1 in the preparation of insecticides.

4. The use according to claim 3, characterized in that: The insecticide targets aphids.

5. The use according to claim 4, characterized in that: The aphid is green peach aphid.

6. An insecticide, characterized in that: The active ingredient comprises the peptide compound according to claim 1.

7. The insecticide according to claim 6, characterized in that The pesticide also includes pesticide adjuvants.

8. The insecticide according to claim 7, characterized in that The pesticide adjuvant includes a dispersant, a cosolvent, a stabilizer, a wetting agent or a spreader.

9. A method for controlling aphids, characterized in that: The method comprises the step of spraying the peptide compound according to claim 1 or the insecticide according to any one of claims 6 to 8 on the plant.

10. The method according to claim 9, characterized in that The effective spraying dose of the peptide compound is ≥0.1 μM.

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