Disinsection fungal engineering strain inverted from scorpion toxin gene and applications

A gene and neurotoxin technology, applied in the field of genetic engineering, can solve the problems of slow insecticidal effect and hindering the large-scale application of microbial pesticides

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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, similar to bacterial or viral insecticides, fungal insecticides also have the weakness of slow insecticidal effects. Under suitable conditions, it generally takes 3-7 days, which hinders the use of fungal insecticides to a large extent. The large-scale application of microbial pesticides including pesticides, especially in the control of explosive pests, microbial pesticides have great limitations

Method used

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  • Disinsection fungal engineering strain inverted from scorpion toxin gene and applications
  • Disinsection fungal engineering strain inverted from scorpion toxin gene and applications
  • Disinsection fungal engineering strain inverted from scorpion toxin gene and applications

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] Embodiment 1 Gene synthesis and vector construction

[0149] 1. Gene construction

[0150] The design of the gene is as follows, the amino acid sequence of the North African scorpion's specific neurotoxin polypeptide AaIT against insects is reverse-coded into a gene of insecticidal fungus preference, and the 5'-end of the gene adds the Metarhizium anisopliae gene MCL1 (see GenBank entry No.: DQ238488 or DQ238489; Or Wang, C.S. and St Leger, R.J.2006.A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses.Proc Natl Acad Sci USA.103:6647-6652) signal peptide (MCL1 SP, amino acid sequence is MRELSSVLAL SGLLALASA) coding sequence and MCL1 mRNA 5'-untranslated region. Moreover, BamHI restriction sites were designed at both ends of the sequence to facilitate cutting and ligation.

[0151] The gene is artificially synthesized by conventional methods, and the synthetic sequence is as follows: figure 1 shown, and clone it into plasmid pUC...

Embodiment 2

[0171] Example 2 fungal transformation and expression verification

[0172] The previously constructed expression vectors pPcGFP and pMcl1prAaIT were digested with SwaI and linearized using fungal protoplasts (see Wang, C.S. and St Leger, R.J. 2006. Acollagenous protective coat enables Metarhizium anisopliae to evadeinsect immune responses.Proc Natl Acad Sci USA .103:6647-6652) for the transformation of Metarhizium anisopliae strain ARSEF 549 (purchased from the USDA Entomogenic Fungal Culture Collection http: / / arsef.fpsnl.cornell.edu / mycology / ARSEF_Culture_Collection.html#Catalog) , screened to obtain genetically stable transformants GFP-549 and AaIT-549.

[0173] 1. Induced expression experiment using Metarhizium anisopliae GFP-549 transformants

[0174] GFP-549 spores were inoculated on potato agar (PDA, Difco Company) medium, cultured at 25°C for 20 days, conidia were collected, and inoculated in Sabouraud’s dextrose nutrient solution (SDB, Difco Company) and basic medium...

Embodiment 3

[0195] The preparation of embodiment 3 pesticide formulations

[0196] On the potato agar (PDA, purchased from Difco company) medium, inoculate AaIT-549 spores, after cultivating at 25 ° C for 20 days, collect conidia, and use 0.05% Tween-20 (Sigma company) to prepare the spore suspension of each bacterial strain liquid.

[0197] Described pesticide preparation formula is as table 1:

[0198] Table 1AaIT-549 spore suspension preparation

[0199] Preparation 1

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Abstract

The invention discloses a polynucleotide sequence of optimized coding Androctonus australis neurotoxin polypeptide AaIT. The polynucleotide sequence is suitable for the transcription expression in fungi. The invention also discloses a fungus strain capable of expressing Androctonus australis neurotoxin polypeptide AaIT as well as a preparation used to control insects, wherein the preparation does not do harm to the environment and other animals and plants except insects, thereby belonging to an environment-friendly preparation. The invention also discloses a promoter used to guide the specific expression of target gene in insect hemocoel. The invention combines well the characteristics of fungus cell that fungus cell can infect insects and parasitize in the bodies of insects and the toxicity of insect toxin polypeptide for the first time, thereby greatly increasing the insect killing efficiency of the fungi.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more particularly, the invention relates to an optimized polynucleotide encoding the North African scorpion neurotoxin polypeptide AaIT, a fungal engineering strain capable of expressing the North African scorpion neurotoxin polypeptide AaIT, and using the strain to kill Insect approach. Background technique [0002] With the development of social economy and the improvement of people's living standards, the research and application of environmentally friendly microbial pesticides have received more and more attention. There are many kinds of microbial pesticides, and more than 2,000 kinds have been found. According to the types of microorganisms, they can be divided into bacteria, fungi, viruses, protozoa and nematodes. At present, there are mainly bacterial insecticides, fungal insecticides, and virus insecticides that have been developed, applied and commercialized. [0003] Fungal ins...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/79A01N63/04A01P7/04C12N15/63A01N63/30A01N63/50
CPCA01N63/00A01N63/02A01N63/04C07K14/43522A01N63/30A01N63/50A01N63/16
Inventor 王成树吕丁丁
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