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Polynucleotide sequences encoding scorpion toxin and transformed insecticidal fungi

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

Inactive Publication Date: 2013-11-20
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • 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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  • Polynucleotide sequences encoding scorpion toxin and transformed insecticidal fungi
  • Polynucleotide sequences encoding scorpion toxin and transformed insecticidal fungi
  • Polynucleotide sequences encoding scorpion toxin and transformed insecticidal fungi

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0164] Embodiment 1 Gene synthesis and vector construction

[0165] 1. Gene construction

[0166]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 (MCL1SP, amino acid sequence 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.

[0167] The gene is artificially synthesized by conventional methods, and the synthetic sequence is as follows: figure 1 shown, and clone it into plasmid pUC57 (G...

Embodiment 2

[0184] Embodiment 2 fungus (metarrhizia) transformation and expression verification

[0185] The expression vectors pPcGFP and pMcl1prAaIT constructed above were digested with SwaI and linearized, followed by the method of using fungal protoplasts (see 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) carried out Metarhizium anisopliae strain ARSEF549 (purchased from the US Department of Agriculture Entomogenic Fungal Culture Collection http: / / arsef.fpsnl.cornell.edu / mycology / ARSEF_Culture_Collection.ht ml#Catalog) Transformation, screening to obtain genetically stable transformants GFP-549 and AaIT-549.

[0186] 1. Induced expression experiment using Metarhizium anisopliae GFP-549 transformants

[0187] 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...

Embodiment 3

[0208] The preparation of embodiment 3 pesticide formulations

[0209] AaIT-549 spores were inoculated on potato agar (PDA, purchased from Difco Company) medium, and after culturing at 25°C for 20 days, conidia were collected, and 0.05% Tween-20 (Sigma Company) was used to prepare the spore suspension of each strain liquid.

[0210] Described pesticide preparation formula is as table 1:

[0211] Table 1AaIT-549 spore suspension preparation

[0212]

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Abstract

Provided are optimized polynucleotide sequences encoding a scorpion Androctonus australis neurotoxin AaIT, which are suitable for being transcribed and expressed in fungi. Also provided are the fungi capable of expressing AaIT, such as Metarhizium anisopliae, which have improved insecticidal efficiency and can be used for insect control in effect.

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 Patents(China)
IPC IPC(8): C12N15/12C12N15/62C12N15/63C12N15/80C12N1/14A01N63/04A01N63/30A01N63/50
CPCA01N63/00A01N63/02A01N63/04C07K14/43522A01N63/30A01N63/50A01N63/16
Inventor 王成树吕丁丁
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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