CN-122012258-A - Metarhizium anisopliae engineering bacteria expressing spider toxic peptide Hv1a and application thereof in preventing and treating chilo suppressalis

CN122012258ACN 122012258 ACN122012258 ACN 122012258ACN-122012258-A

Abstract

The invention belongs to the technical field of biological control of agricultural pests, and particularly relates to a metarhizium anisopliae engineering bacterium expressing spider toxic peptide Hv1a and application thereof in control of chilo suppressalis. The invention discloses the remarkable insecticidal activity of metarhizium anisopliae (Metarhizium guizhouense) ARSEF 977 on the chilo suppressalis for the first time, and based on the remarkable insecticidal activity, the Hv1a toxic peptide gene derived from the spider is introduced into the strain by a genetic engineering technology, so that an engineering strain for stably and efficiently expressing the insecticidal peptide Hv1a of the spider is successfully constructed, the engineering strain plays a synergistic effect, the insecticidal effect on the chilo suppressalis is further improved, and the death rate after 7 days of treatment is as high as 96.7 percent, and can be used for the control of the chilo suppressalis. The invention provides a high-potential starting strain for developing a special fungal pesticide for the chilo suppressalis, and provides a novel material and a novel method for promoting the development of a green high-efficiency prevention and control technology of the chilo suppressalis, and has a wide application prospect.

Inventors

HE YUEPING
CAI QING
Jiao Xingxia
Cai Chuanlai
ZHANG YIRAN
WANG YONGMO
ZHAO JING

Assignees

华中农业大学

Dates

Publication Date: 20260512
Application Date: 20260205

Claims (10)

1. The engineering bacteria are engineering bacteria for expressing the spider venom peptide Hv1a, and are characterized in that the engineering bacteria are engineering bacteria for transferring the spider venom peptide Hv1a gene into the green muscardine fungus so as to express the spider venom peptide Hv1a, wherein the amino acid sequence of the spider venom peptide Hv1a is shown as SEQ ID NO.2, and the green muscardine fungus is green muscardine fungus (Metarhizium guizhouense) ARSEF 977.
2. The engineering bacterium according to claim 1, wherein the nucleotide sequence of the spider toxic peptide Hv1a gene is shown in SEQ ID NO. 1.
3. The engineering bacterium according to claim 1, wherein the preparation method of the metarhizium anisopliae engineering bacterium for expressing the spider venom peptide Hv1a comprises the steps of connecting a Hv1a toxic peptide gene to a pCAMBIA0380 expression vector, transforming the recombinant vector into metarhizium anisopliae by an agrobacterium-mediated genetic transformation method, and screening to obtain the recombinant engineering bacterium for completely expressing the spider venom peptide Hv1 a.
4. The application of the metarhizium anisopliae and/or the metarhizium anisopliae engineering bacteria expressing the spider venom peptide Hv1a in any one of the following: A1 Application in preventing and treating chilo suppressalis; a2 Application in preparing products for preventing and treating chilo suppressalis; The amino acid sequence of the spider venom peptide Hv1a is shown as SEQ ID NO.2, and the metarhizium anisopliae is metarhizium anisopliae (Metarhizium guizhouense) ARSEF 977.
5. A method for preventing and controlling chilo suppressalis is characterized by comprising the steps of treating chilo suppressalis by using metarhizium anisopliae and/or metarhizium anisopliae engineering bacteria expressing spider poison peptide Hv1a, wherein the amino acid sequence of the spider poison peptide Hv1a is shown as SEQ ID No.2, and the metarhizium anisopliae is metarhizium anisopliae (Metarhizium guizhouense) ARSEF 977.
6. The method according to claim 5, wherein the metarhizium anisopliae and/or metarhizium anisopliae engineering bacteria expressing the spider venom peptide Hv1a are prepared into spore suspension, the chilo suppressalis is subjected to insect dipping treatment, and/or the chilo suppressalis is subjected to spraying treatment on plants or leaves or soil of harmful crops.
7. The method of claim 6, wherein the spore suspension has a concentration of not less than 2.00 x 10 7 spores/ml.
8. The method according to claim 5, wherein the engineering strain of metarhizium anisopliae expressing the spider venom peptide Hv1a is produced by transferring the Hv1a toxic peptide gene shown in SEQ ID NO.1 into metarhizium anisopliae so as to express the spider venom peptide Hv1a.
9. The method of claim 8, wherein the preparation method of the metarhizium anisopliae engineering bacteria for expressing the spider venom peptide Hv1a comprises the steps of connecting the Hv1a toxic peptide gene to a pCAMBIA0380 expression vector, transforming the recombinant vector into metarhizium anisopliae by an agrobacterium-mediated genetic transformation method, and screening to obtain the recombinant engineering bacteria for completely expressing the spider venom peptide Hv1 a.
10. The method according to claim 9, wherein the method for preparing the engineering strain of metarhizium anisopliae expressing the spider venom peptide Hv1a comprises the following steps: S1, constructing an expression vector, namely sequentially connecting a signal peptide coding gene, an Hv1a toxic peptide gene and a FLAG protein coding gene to form an Hv1a-FLAG fusion expression element, and inserting the Hv1a-FLAG fusion expression element into the middle of a Ptef1 promoter and a sur selection marker of a pCAMBIA0380 expression vector to obtain a p0380-Ptef1-Hv1a-FLAG-sur heterologous expression vector; s2, fungus genetic transformation, namely introducing a p0380-Ptef1-Hv1a-FLAG-sur heterologous expression vector into metarhizium anisopliae by an agrobacterium-mediated genetic transformation method; S3, screening and identifying engineering bacteria, namely screening positive transformants in M-100 medium containing chlorimuron-ethyl Long Kangsheng by using sur screening markers on the expression vector, and verifying integration of Hv1a genes by PCR amplification to obtain the metarhizium anisopliae engineering bacteria expressing the spider venom peptide Hv1 a.

Description

Metarhizium anisopliae engineering bacteria expressing spider toxic peptide Hv1a and application thereof in preventing and treating chilo suppressalis Technical Field The invention belongs to the technical field of biological control, and particularly relates to a metarhizium anisopliae engineering bacterium for expressing spider venom peptide Hv1a and application thereof in control of chilo suppressalis. Background Chilo suppressalis (Chilo suppressalis) is one of the most difficult main pests to control on rice, belongs to boring pests, and the larvae of the rice stem boring pests drill into the rice stem to eat, so that symptoms such as dead centers, white ears and the like are caused, and the rice is greatly reduced in yield and even is out of harvest when serious. At present, the control of the chilo suppressalis still highly depends on chemical pesticides, and the long-term massive use of the chilo suppressalis not only leads to the increasing of the drug resistance of pests, but also causes the problems of pesticide residues, ecological environment pollution, quality safety of agricultural products and the like. Therefore, development of novel efficient green prevention and control products and technologies is urgent. Microbial pesticides are important categories of biopesticides, and are hot spots for research and application in recent years because they can kill pests by means of infection, parasitism, or active substance generation. The single-dose microbial pesticide products for preventing and treating the chilo suppressalis only comprise bacillus thuringiensis (Bacillus thuringiensis, bt), beauveria bassiana (Beauveria bassiana) and metarhizium anisopliae (Metarhizium anisopliae). The microbial pesticide product has the advantages of safety, relatively difficult resistance generation and the like in the aspect of the control of the chilo suppressalis, but also has the common problems of low insecticidal speed, large influence of environmental conditions on the mortality, unstable control effect and the like, and is difficult to completely meet the high-efficiency and quick-acting control demands in the field. Genetic improvement of biocontrol bacteria by genetic engineering means has been explored in order to overcome the limitations of natural microbial pesticides. There are studies on the attempt of introducing exogenous toxin genes, insecticidal protein genes and the like into fungal vectors such as beauveria bassiana, metarhizium anisopliae and the like to enhance the insecticidal toxicity and the action speed, wherein the spider venom peptide Hv1a is derived from Australian funnel net spiders, is neurotoxin which specifically acts on voltage-gated calcium channels of insects, has strong toxicity to various insects and has higher safety to mammals, so that the spider venom peptide Hv1a has the potential of being developed into a novel biological insecticide. However, construction of the engineering bacteria still faces multiple challenges such as exogenous gene expression efficiency, engineering bacteria genetic stability, field adaptability and safety evaluation. At present, high-efficiency engineering fungus preparations for Chilo suppressalis are still lacking. Disclosure of Invention Aiming at the blank of the prior art, the invention successfully constructs an engineering strain for stably and efficiently expressing the spider insecticidal peptide Hv1a by introducing the Hv1a toxic peptide gene derived from the spider into the Metarrhizium anisopliae strain through a genetic engineering technology on the basis of finding out a strain of Metarrhizium anisopliae (Metarhizium guizhouense) with higher insecticidal activity on the chilo suppressalis, and finds out that the engineering strain shows excellent insecticidal effect on the chilo suppressalis, can be used for preventing and controlling the chilo suppressalis, provides a novel material and a novel method for promoting the development of green pest prevention and control technology, and has wide application prospect. The invention aims at providing a metarhizium anisopliae engineering bacterium for expressing a spider venom peptide Hv1a, wherein the engineering bacterium is used for transferring a spider venom peptide Hv1a gene into metarhizium anisopliae so as to enable the spider venom peptide Hv1a to be stably expressed and secreted, the amino acid sequence of the spider venom peptide Hv1a is shown as SEQ ID No.2, and the metarhizium anisopliae is metarhizium anisopliae (Metarhizium guizhouense) ARSEF 977. Further, the nucleotide sequence of the spider venom peptide Hv1a gene is shown as SEQ ID NO. 1. Further, the preparation method of the metarhizium anisopliae engineering bacteria for expressing the spider venom peptide Hv1a comprises the steps of connecting the Hv1a toxic peptide gene to a pCAMBIA0380 expression vector, transforming the recombinant vector into metarhizium anisopliae by an agrobacterium-mediated genetic transfo