CN116410291A - Bug polypeptide Ple1 and application thereof - Google Patents
Bug polypeptide Ple1 and application thereof Download PDFInfo
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- CN116410291A CN116410291A CN202310386682.3A CN202310386682A CN116410291A CN 116410291 A CN116410291 A CN 116410291A CN 202310386682 A CN202310386682 A CN 202310386682A CN 116410291 A CN116410291 A CN 116410291A
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Images
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a beneficial stinkbug polypeptide Ple and application thereof, belonging to the technical field of biological control, wherein the full-length sequence of a coding sequence region of a beneficial stinkbug polypeptide Ple1 gene is ATGAAGTTCTATCTCCTTCTGTTCCTCGTCATCGTCGTCCTCGCCTGCGGGGTCCTGGCTCTTCAAGAGCCCAACTGCATACAGAGAGGAAAGGAATGCGTTGGCGCATCCATGGGATGCTGTAAGGGCTTGAGGTGCCTGTACTACGCCAACAGGTGTGTTGGGCCGTGA, a mature peptide sequence is LQEPNCIQRGKECIGASMGCCKGLRCLYYANRCVGP, 6 cysteines form 3 disulfide bonds, and the disulfide bonds are C1-C4, C2-C5 and C3-C6. The beneficial stink bug polypeptide Ple1 has insecticidal activity on myzus persicae, prodenia litura and chilo suppressalis.
Description
Technical Field
The invention belongs to the technical field of biological control, and particularly relates to a beneficial stinkbug polypeptide and application of the beneficial stinkbug polypeptide in preparing a tobacco aphid, a prodenia litura and a chilo suppressalis pesticide.
Background
Biological pesticides are important points in the current pesticide development because of their abundant natural resources, low production cost and relatively good environmental compatibility. Animal toxins are an important source of efficient pesticide development, particularly toxins of natural enemies of insects, have target specificity more than traditional pesticides, and are a good resource for efficient and safe pesticide development (King and Hardy, 2013; senji Laxmeet al, 2019). The insect natural enemy toxin most studied in pesticide development is the spider toxin (King and Hardy, 2013; luddecke et al, 2022). Over ten million insecticidal toxin substances (luddecke et al 2022) are estimated to be present in all existing spider venom throughout the world, but only a few spider toxins have high insecticidal selectivity. For example, the only currently marketed insecticide derived from spider venom is the polypeptide toxin GS-omega/kappa-hexatoxin-Hv 1a based on Australian funnel-shaped spider Hadronyche versuta, which is highly effective against pests while being safe against bees and vertebrates.
Predatory insects have narrower feeding habits and higher selectivity for toxins than other natural enemies such as spiders. At present, predatory insects with more toxin researches comprise predatory bugs, parasitic bees, predatory flies and the like. Predatory bug venom is known to have strong insecticidal activity. Such as stinkbug venom, is infused into insects resulting in paralysis, tissue liquefaction and death (Walker et al, 2016); LD of sample extracted from kidney bug Picromerus nigrispinus poison gland on soybean noctuid 50 About 2 uL/head (Martinez et al 2016).
The most ubiquitous toxin in animal venom is Knottin toxin (King, 2019; matsubara et al, 2017). Knottin is a small peptide having a kinked structure (Knottin) of 3 pairs or more of disulfide bonds, and is very stable in an organism. Knottin polypeptide structure has remarkable plasticity, and different organisms evolved that a rich variety of Knottin polypeptides perform different functions (King, 2019). For example, the commercial spider poison pesticide active ingredient GS-omega/kappa-hexatoxin-Hv 1a is a Knottin polypeptide. Knottin toxin in the venom of predator bugs has activity in inhibiting voltage-gated calcium ion channels (cav 2.2), but it has not been found to have insecticidal activity (Corzo et al, 2001; rugen et al, 2021).
The plant bug Picromerus lewisi (Scott) which is the predatory bug can predate more than 40 agricultural and forest pests such as lepidoptera and coleoptera, such as prodenia litura, spodoptera frugiperda, phyllostachys praecox and the like, is an excellent predatory natural enemy, and has been artificially bred and applied in large scale at present. However, no research report on the stinkbug venom and toxins is currently reported. Since the beneficial stink bug is always in the field ecosystem and is highly homologous to the plant feeding stink bug in evolution, the beneficial stink bug toxin may have high selectivity, so that the development of knottin toxic peptide with insecticidal activity based on the beneficial stink bug venom is more suitable for green prevention and control of pests.
Disclosure of Invention
The invention aims to overcome the defects and provide the beneficial stinkbug polypeptide Ple1 with insecticidal activity on myzus persicae, prodenia litura and chilo suppressalis.
The invention further aims at providing application of the beneficial stink bug polypeptide Ple1 in preparing pesticide of myzus persicae, prodenia litura and chilo suppressalis.
The full-length sequence of the coding sequence region (CDS) of the beneficial stinkbug polypeptide Ple1 gene is ATGAAGTTCTATCTCCTTCTGTTCCTCGTCATCGTCGTCCTCGCCTGCGGGGTCCTGGCTCTTCAAGAGCCCAACTGCATACAGAGAGGAAAGGAATGCGTTGGCGCATCCATGGGATGCTGTAAGGGCTTGAGGTGCCTGTACTACGCCAACAGGTGTGTTGGGCCGTGA, the mature peptide sequence is LQEPNCIQRGKECIGASMGCCKGLRCLYYANRCVGP, and 3 disulfide bonds are formed by 6 cysteines, and the disulfide bonds are C1-C4, C2-C5 and C3-C6.
The application of the beneficial stink bug polypeptide Ple1 in preparing pesticide of myzus persicae, prodenia litura and chilo suppressalis.
Compared with the prior art, the invention has obvious beneficial effects, and the technical scheme can be adopted as follows: the insecticidal activity of the beneficial stink bug polypeptide on the two-instar nymphs of the myzus persicae is measured by an artificial feed feeding method, and the mortality rate of the Ple polypeptide 10 mu mol/L treated for 5 days is 86.7%, which shows that the tested concentration of the beneficial stink bug Ple1 polypeptide has higher poisoning activity on the myzus persicae. The insecticidal activity of the beneficial stink bug polypeptide on the second-instar larvae of the prodenia litura is measured by a microinjection method, and the mortality rate of 72 hours of treatment with the Ple1 polypeptide concentration of 10 mu mol/L is 66.7%, which shows that the Ple polypeptide test concentration has higher toxicity on the prodenia litura. The insecticidal activity of the beneficial stink bug polypeptide on the four-instar larvae is measured by a microinjection method, and the mortality rate of the Ple polypeptide after being treated by 40 mu mol/L for 7d of the chilo suppressalis is found to be 80%, which indicates that the tested concentration of the beneficial stink bug Ple polypeptide has higher toxicity on the chilo suppressalis.
Drawings
FIG. 1 is a sequence alignment analysis of the beneficial bug polypeptide Ple1, the hunter bug toxin Ptu and the spider toxin Hv1a (Ptu 1: heidelus wuziensis, P58606.1, hv1a: funnel net spider, P56207);
FIG. 2 is a three-dimensional structure of a beneficial bug polypeptide Ple1 (homology modeling using SWISS-MODEL online website, template lmr.1.A (taxi ADO 1), with the dark color indicating cysteine);
FIG. 3 is a mass spectrometric detection diagram of a sample prepared by chemical synthesis of a beneficial bug polypeptide Ple1
FIG. 4 is a diagram of HPLC analysis of a chemically synthesized preparation sample of beneficial bug polypeptide Ple1
FIG. 5 shows the virulence of beneficial bug polypeptide Ple1 against Aphis tabaci (Artificial feeding, positive control is abamectin 8.87 mg/L)
FIG. 6 shows virulence of beneficial bug polypeptide Ple1 against Spodoptera litura (injection, positive control is abamectin 0.89 ng/head)
FIG. 7 shows the virulence of the beneficial bug polypeptide Ple1 against Chilo suppressalis (injection)
Detailed Description
Example 1: a method for synthesizing a beneficial bug polypeptide Ple1, comprising the steps of:
(1) Method for designing and constructing polypide gene of orius sinensis
Dissecting the adult human stinkbug gland 50 pairs, extracting total RNA, and sending three repeated samples to a sequencing company for RNA-seq transcriptome sequencing analysis. The beneficial bug polypeptide genes were found based on annotation information and BLAST. Extracting total RNA of adult plant bug, reverse transcribing to obtain cDNA template, and PCR amplifying target gene to perform sequencing verification.
In the transcriptome data of the beneficial stinkbug gland, a small peptide gene rich in cysteine was found and named as beneficial stinkbug polypeptide Ple1.
In the transcriptome data of the armyworm, a small peptide gene rich in cysteine was named as the beneficial stinkbug polypeptide Ple1. The full-length sequence of the coding sequence region (CDS) of the beneficial stinkbug polypeptide Ple1 gene is ATGAAGTTCTATCTCCTTCTGTTCCTCGTCATCGTCGTCCTCGCCTGCGGGGTCCTGGCTCTTCAAGAGCCCAACTGCATACAGAGAGGAAAGGAATGCGTTGGCGCATCCATGGGATGCTGTAAGGGCTTGAGGTGCCTGTACTACGCCAACAGGTGTGTTGGGCCGTGA through gene clone sequencing verification. The mature peptide sequence of the beneficial bug polypeptide Ple is LQEPNCIQRGKECIGASMGCCKGLRCLYYANRCVGP, and like the known stinkbug toxin Ptu1 and spider toxin Hv1a, contains 6 cysteines to form 3 disulfide bonds in a manner of C1-C4, C2-C5, and C3-C6, but with a low amino acid sequence similarity (25% -35%) (FIG. 1). The sequence of the toxic peptide gene is verified to be correct by gene clone sequencing.
(2) Chemical synthesis of beneficial bug polypeptide Ple1
The beneficial stinkbug Ple polypeptide (LQEPNCIQRGKECIGASMGCCKGLRCLYYANRCVGP) has an active ingredient content of 93.78% and is produced by the blaze biotechnology company, inc. through 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemical synthesis technology. The disulfide bond orientation is ensured, i.e. the first and fourth, second and fifth, third and sixth cysteines at the N-terminus form disulfide bonds (C1-C4, C2-C5 and C3-C6), respectively.
The synthesis steps are as follows: connecting Fmoc-protected C-terminal first amino acid with resin; removing Fmoc protecting groups, and detecting whether the connection is successful or not by ninhydrin; adding a second Fmoc protected amino acid for condensation, and detecting ninhydrin; then sequentially adding different amino acids for connection until the last amino acid; finally adding cutting fluid to cut Fmoc and resin; adding diethyl ether, centrifuging and drying; modifying the directional disulfide bond by using a DMSO oxidation method to obtain a crude product; separating and purifying by High Performance Liquid Chromatography (HPLC), and lyophilizing to obtain the final product. Finally, mass spectrometry confirmation (fig. 3) and HPLC purity identification (fig. 4) were performed.
Test example 1: determination of insecticidal Activity of beneficial bug polypeptide Ple1 against Aphis tabaci
The insecticidal activity of the beneficial stinkbug polypeptide on the two-instar nymphs of myzus persicae is determined by adopting an artificial feed feeding method. The polypeptide is prepared into a polypeptide mother solution with the concentration of 1mmol/L by DMSO as it is, and then diluted into a test concentration solution by artificial feed. An artificial feed solution of 1% dmso was used as a control. The treated test insects were transferred to a petri dish (10 cm diameter dish bottom poured into 2% agar) containing tobacco leaf dishes. The artificial feed was changed every other day, 10 replicates each, 3 replicates. The dishes were placed in a culture chamber at a temperature of 21.+ -. 1 ℃, a humidity of 50%.+ -. 10% and an illumination period of 16:8 (L: D) h.
The 5-day mortality rate of the solvent control group in the test was 30%. The 5-day mortality of Ple polypeptide 10. Mu. Mol/L was 86.7% (FIG. 5). The result shows that the tested concentration of the beneficial stinkbug Ple1 polypeptide has higher poisoning activity on the myzus persicae.
Test example 2: determination of insecticidal activity of beneficial stinkbug polypeptide Ple1 on prodenia litura
The insecticidal activity of the beneficial stinkbug polypeptide on the second instar larvae of the prodenia litura is measured by a microinjection method. And (3) respectively injecting different toxic peptide solutions into the bodies of the 2-year-old prodenia litura larvae by using a microinjection instrument, wherein the injection part is the midchest back part of the prodenia litura larvae. As a control, an aqueous solution containing 1% DMSO was used. The injection quantity per head is 100nL, and the injection flow rate is 20nL/s. Each injection treatment test insect was transferred to a petri dish (10 cm diameter dish bottom poured into 2% agar) containing tobacco leaf discs. 10 heads per treatment, three replicates for 30 heads. The temperature is 27+ -1deg.C, the humidity is 50% + -10%, and the illumination period is 16:8 (L: D) h. The death of the test insects was observed 24h, 48h and 72h after injection. The death symptom judgment standard is that the brush pen has no autonomous response when touched.
Mortality within 72h of the test control group was no more than 20%. Ple1 polypeptide concentration 10. Mu. Mol/L mortality was 66.7% in 72h treatment (FIG. 6). The result shows that the Ple polypeptide test concentration has higher toxicity to prodenia litura.
Test example 3: determination of insecticidal activity of beneficial stinkbug polypeptide Ple1 on chilo suppressalis
The insecticidal activity of the beneficial stinkbug polypeptide on the four-instar larvae of chilo suppressalis is measured by a microinjection method. And injecting the test polypeptide solution into the bodies of 4-year-old Chilo suppressalis larvae by using a microinjection instrument, wherein the injection part is the internode membranes of the second section and the third section of the ventral surface of the Chilo suppressalis larvae. As solvent control, an aqueous solution containing 4% dmso was used. The injection quantity per head is 100nL, and the injection flow rate is 20nL/s. The injected Chilo suppressalis larva is transferred into a conical plastic small box (the diameter of the bottom is 3cm, the diameter of the top is 3.7cm, the height is 3.3 cm), a proper amount of artificial feed is placed at the bottom of the box, and the top is pricked with small Kong Fangbian Chilo suppressalis and breathable). A total of 10 heads per treatment. The dishes were placed in a culture chamber at a temperature of 27.+ -. 1 ℃, a humidity of 70.+ -. 10% and a light dark period of 16h:8 h. Death was observed daily after injection. The death symptom judgment standard is that the brush pen has no autonomous response when touched.
Mortality of the solvent control group 7d was 20% and mortality of the Ple polypeptide 40. Mu. Mol/L treated chilo suppressalis 7d was 80% (FIG. 7). The result shows that the testing concentration of the beneficial stinkbug Ple1 polypeptide has higher toxicity to the Chilo suppressalis.
Through a bioassay experiment, the beneficial stink bug polypeptide Ple has higher toxicity to the myzus persicae, the prodenia litura and the chilo suppressalis.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any simple modification, equivalent variation and variation of the above embodiment according to the technical matter of the present invention still fall within the scope of the technical scheme of the present invention.
Claims (3)
1.A beneficial bug polypeptide Ple, the full length sequence of the coding sequence region of the gene of which is ATGAAGTTCTATCTCCTTCTGTTCCTCGTCATCGTCGTCCTCGCCTGCGGGGTCCTGGCTCTTCAAGAGCCCAACTGCATACAGAGAGGAAAGGAATGCGTTGGCGCATCCATGGGATGCTGTAAGGGCTTGAGGTGCCTGTACTACGCCAACAGGTGTGTTGGGCCGTGA.
2. The beneficial bug polypeptide Ple1 of claim 1 having a mature peptide sequence LQEPNCIQRGKECIGASMGCCKGLRCLYYANRCVGP with 6 cysteines forming 3 disulfide bonds in a manner of C1-C4, C2-C5, C3-C6.
3. Application of beneficial stink bug polypeptide Ple in preparing pesticide of myzus persicae, prodenia litura and chilo suppressalis.
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| CN116789787A (en) * | 2023-03-24 | 2023-09-22 | 贵州省烟草公司遵义市公司 | Isoma sinensis polypeptide Ach1 and application thereof |
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2023
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|---|---|---|---|---|
| US5554592A (en) * | 1992-03-04 | 1996-09-10 | Sandoz Ltd. | Insecticidal toxins from the parastic wasp, bracon hebetor |
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| Title |
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