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Chipper borer carboxylesterase and its coding gene, preparation method and application

A technology of chilo carboxylate and chilo carboxylic acid, applied in the field of genetic bioengineering, can solve the problems of lack of sensitivity, selectivity and stability of acetylcholinesterase species, and limitations of commercial application

Active Publication Date: 2016-04-13
ZHEJIANG UNIV
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Problems solved by technology

Acetylcholinesterase is combined with OPs and CBs pesticides to detect pesticide residues, but this type of detection method is often affected by the lack of acetylcholinesterase species, as well as sensitivity, selectivity and stability issues, which ultimately lead to commercial applications. limit

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  • Chipper borer carboxylesterase and its coding gene, preparation method and application
  • Chipper borer carboxylesterase and its coding gene, preparation method and application
  • Chipper borer carboxylesterase and its coding gene, preparation method and application

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Embodiment 1

[0026] Use Trizol reagent to extract total RNA from the 4th instar larvae of Chilo suppressalis, and synthesize cDNA through reverse transcriptase reaction; design specific primers containing enzyme cleavage sites, and use high-fidelity DNA polymerase to amplify the synthesized cDNA as a template The complete open reading frame carboxylesterase gene of Chilo borer with restriction sites at the 2 ends was digested with BamHI and XhoI, and then ligated into the transfer vector pFastBac-HTB that had been cut with the same restriction enzymes.

[0027] The primer sequences are as follows:

[0028] F_CsCOE026-BamHI: 5'-CGGGATCCATGTTCGAATATTTG-3';

[0029] R_CsCOE026-XhoI: 5'-CCGCTCGAGTTATTTTCTTTATTGT-3'.

[0030] The base sequence of the C. borer carboxylesterase gene CsCOE026 is shown in SEQ ID No.1.

[0031] The recombinant transfer vector was transformed into Escherichia coli DH10Bac competent cells, and plated on LB plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10...

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Abstract

The invention discloses chilo suppressalis carboxylesterase as well as a coding gene, preparation method and application of chilo suppressalis carboxylesterase. An amino acid sequence of chilo suppressalis carboxylesterase is shown in SEQ ID No.2; a chilo suppressalis carboxylesterase gene is shown in SEQ ID NO.1; the chilo suppressalis carboxylesterase gene is cloned from 4-year-old chilo suppressalis larvae, recombinant viruses are quickly generated through a Bac-to-Bac baculovirus expression system, and high-expression and high-activity chilo suppressalis carboxylesterase is successfully obtained from insect cells. Chilo suppressalis carboxylesterase can be applied to detection and degradation of pesticides, the problem that less types of insect acetylcholin esterase varieties are available at present is solved, and a new pesticide detection way is provided.

Description

technical field [0001] The present invention relates to the field of genetic bioengineering, in particular to a rice stem borer carboxylesterase and its coding gene, preparation method and application. Background technique [0002] Pesticides are a class of biologically toxic chemical substances that humans actively put into the environment in large quantities. At present, the problem of excessive use of pesticides in my country is very serious. Only about 1% of the pesticides used in large quantities can exert their efficacy, and most of the rest remain in the soil, float in the atmosphere or enter the waters through runoff, seriously endangering the ecological environment and human food safety. damage to human health. Pesticide residue pollution of agricultural products in my country is very common and quite serious, and the phenomenon of acute and chronic poisoning caused by pesticides and their residue pollution among Chinese people is relatively common. In addition, th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/866C12Q1/44A62D3/02A62D101/04A62D101/26A62D101/28
CPCC12N9/18C12N15/86C12N2710/14043C12Y301/01001
Inventor 姚洪渭巨青松叶恭银
Owner ZHEJIANG UNIV
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