Insecticidal active polypeptide and application thereof

Abstract

The invention discloses an insecticidal active polypeptide and application thereof, and relates to the technical field of biology. According to the present invention, the structure of the insect neuropeptide Kinin is modified, and computer-aided design is combined to develop the insecticidal active polypeptide R01, such that the new prevention and control idea is provided for the biological prevention and control of aphids. The insecticidal active polypeptide R01 is not only suitable for prevention and control of aphids, but also can play a role in pest management in agriculture and other fields, and has wide application potential. The insecticidal active polypeptide R01 designed by the invention can influence the function exertion of neuropeptide receptors of pests and further influence the growth and development of the pests, so that the control on pest populations is realized, and compared with traditional pesticides, the insecticidal active polypeptide R01 has a lower non-target effect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an insecticidal active polypeptide and application thereof. Background Art

[0002] Aphids belong to the order Hemiptera and include two major classes: Adelgoide and Aphidoidea. Aphids harm 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 vectors 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 capacity, can reproduce sexually and parthenogenetically, have a short generation cycle, a large number, and a very serious phenomenon of generational replacement. Therefore, aphid control is quite difficult.

[0003] Chemical control is currently the main method of controlling aphids. Although chemical control is highly effective and easy to operate, chemical pesticide residues lead to ecological damage and soil pollution. In addition, excessive use of chemical pesticides leads to pest resistance, which hinders the development of agricultural and forestry production in my country. Therefore, the development of a green, safe and highly targeted biological pesticide is a new idea of ​​great significance.

[0004] As a natural biologically active substance, insect neuropeptides are characterized by high efficiency in trace amounts and strong targeting, and can regulate various physiological functions of insects. Neuropeptides target their specific receptors, and the raw materials are natural amino acids, which are suitable targets for the development of green biological pesticides. The inventor's research group has been conducting research on insect neuropeptides for a long time, and has a good foundation for the development of neuropeptide analogs of green biological pesticides. Research results show 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 analog with aphid-killing activity. Summary of the invention

[0005] The purpose of the present invention is to provide an insecticidal polypeptide and its application to solve the problems existing in the above-mentioned prior art. The insecticidal polypeptide can affect the function of the neuropeptide receptors of pests, thereby affecting their growth and development, thereby achieving control of pest populations, and has lower non-target effects than traditional pesticides.

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

[0007] The present invention provides an insecticidal active polypeptide, the structural formula of which is as follows:

[0008]

[0009] The present invention also provides a method for preparing the above insecticidal active polypeptide, comprising the step of synthesizing the insecticidal active polypeptide by solid phase synthesis.

[0010] The present invention also provides the use of the above insecticidal active polypeptide 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 insecticidal active polypeptide.

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

[0015] Furthermore, the pesticide adjuvant includes but is not limited to a dispersant, a solubilizer, 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 insecticidal active polypeptide or insecticide on plant plants.

[0017] The effective spraying dosage of the insecticidal active polypeptide is ≥0.1 μM.

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

[0019] The present invention structurally transforms the insect neuropeptide Kinin and combines it with computer-aided design to develop an insecticidal active polypeptide R01, which provides a new control idea for biological control of aphids. The insecticidal active polypeptide R01 is not only suitable for the control of aphids, but may also play a role in pest management in agriculture and other fields, and has a wide range of application potentials.

[0020] The insecticidal active polypeptide R01 designed by the present invention can affect the function of the neuropeptide receptors of pests, thereby affecting their growth and development, thereby achieving control of the pest population, and has lower non-target effects compared to traditional pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] Figure 1The amino acid structure of the lead compound, natural insect neuropeptide Kinin;

[0023] Figure 2 This is a schematic diagram of the modification of the natural insect neuropeptide Kinin by the present invention;

[0024] Figure 3 is a schematic diagram of the structure of compound R01;

[0025] Figure 4 This is a statistical graph of the direct contact toxicity test results of compound R01 against green peach aphid. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1

[0032] 1. Modification of natural insect neuropeptide Kinin

[0033] (1) The website http: / / stagbeetle.animal.uiuc.edu / cgi-bin / neuropred.py was used to search for the sequence of the natural Myzus persicae neuropeptide Kinin. Its sequence is FSPWG (single letter abbreviation amino acid). The amino acid structure diagram is shown in Figure 1 .

[0034] (2) Use computer software ChemDraw 20.0 to draw the Kinin structure and export it as a mol2 format file.

[0035] (3) Using the NCBI database, we found that the sequence number of the peach aphid Kinin neuropeptide receptor is >XP_003241610.1.

[0036] (4) The Kinin neuropeptide receptor sequence obtained above was homologously modeled using the online website Uniprot to obtain the PDB file of the receptor protein.

[0037] (5) Computer software Discovery Studio 2019 was used to perform computer molecular docking between the ligand mol2 file and its receptor PDB file, using the CDOCKer function. It was found that the second serine in the key binding site did not have a strong force link, so this site was modified.

[0038] (6) Introducing long-chain hydroxyl groups, cycloalkanes, and hybrid groups, 27 new compounds were obtained ( Figure 2 ).

[0039] 2. Activity prediction of unknown active compounds

[0040] (1) 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 with 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.

[0041] (2) The 27 new compounds designed by the present invention were imported into the software SYBYL-X2.0 to perform molecular skeleton model superposition. The prediction model was used to perform 3D-QSAR activity prediction on the 27 unknown active compounds designed by the present invention. It was found that R01 (structural formula see Figure 3 ) has a higher activity value.

[0042] Example 2

[0043] Synthesis of compound R01:

[0044] Compound R01 was synthesized using SPPS solid phase synthesis. Using Fmoc chemistry, amino acids were sequentially added to the resin to build the peptide chain. After the synthesis was completed, the N-terminal Fmoc group was first deprotected, followed by the side chain protecting groups, and the peptide was cleaved from the resin. The specific procedure is as follows:

[0045] 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. The raw material of the non-natural amino acid with 3 carbon cycloalkane at position 2 was purchased from Anaiji Chemical Reagent Network and its name is L-cyclopropylalanine (CAS: 102735-53-5).

[0046] 2. Add pip / DMF solution for a while and then remove the solvent by vacuum filtration.

[0047] 3. Washing after Fmoc removal: Add DMF to the resin (washing step) and then remove the solvent by vacuum filtration.

[0048] 4. QC test resin. The actual MW relative molecular weight is 572.3, which is consistent with the theoretical value.

[0049] Example 3

[0050] Compound R01 synthesized in Example 2 was subjected to direct contact toxicity test on aphids:

[0051] 1. Take 1 mg of compound R01 and dissolve it in 500 μL of dimethyl sulfoxide (DMSO), then add 1247 μL of HPLC water (containing 0.01% Triton) to dilute the solution to 1 mM concentration, and further dilute it to 1 μM concentration with HPLC water (containing 0.01% Triton) to obtain R01 solution for biological assay. Take 1 mg of neuropeptide Kinin and dissolve it in 500 μL of dimethyl sulfoxide (DMSO), then add 1247 μL of HPLC water (containing 0.01% Triton) to dilute the solution to 1 mM concentration, and further dilute it to 1 μM concentration with HPLC water (containing 0.01% Triton) to obtain Kinin solution for biological assay.

[0052] 2. Pick fresh tobacco leaves and cut them into a size suitable for a plastic bowl with a diameter of 10 cm. Soak a sponge in water and put it in the plastic bowl, then put the tobacco leaves in the plastic bowl so that they touch the wet sponge and keep the leaves moist.

[0053] 3. Adult peach aphids were selected for toxicity determination. Twenty aphids in each group were inoculated on the back of tobacco leaves. The experiment was repeated for five groups. 100 nL of solution (1 μM R01 solution or 1 μM Kinin solution) was dripped on each aphid. After the absorption was observed, the aphids were placed in a climate chamber at 25°C and 60% humidity. The number of deaths was observed every day and recorded for 7 days. The same dose of DMSO HPLC aqueous solution (containing 0.01% Triton) was dripped on each aphid in the control group (CK).

[0054] The aphid survival curves obtained by each group are as follows Figure 4 It can be observed that the number of deaths in the R01 experimental group was significantly higher than that in the control group, and there was a very significant difference between the two groups.

[0055] 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. An insecticidal active polypeptide, characterized in that: The structural formula is as follows:

2. A method for preparing the insecticidal polypeptide according to claim 1, characterized in that: The method comprises the step of synthesizing the insecticidal active polypeptide by a solid phase synthesis method.

3. Use of the insecticidal polypeptide according to claim 1 in the preparation of insecticides.

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

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 insecticidal active polypeptide 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 insecticidal polypeptide 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 dosage of the insecticidal active polypeptide is ≥0.1 μM.

Images