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Novel Bt protein Cry53Ab1, coding gene thereof and use

A technology of bt protein and gene, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2010-08-04
SICHUAN AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, studies have proved that Bti has not yet found resistance problems in the use of field (Regis L, et al., 2000. The use of bacterial larvicides in mosquito and black fly control programs in Brazil. Mem. Instituto Oswaldo Cruz, 95: 207-210. ), but the problem of mosquito resistance to it has been continuously confirmed in the laboratory, and this situation may also appear in the field (Georghiou GP, and Wirth MC, 1997.Influence of exposure to singleversus multiple toxins of Bacillus thuringiensis subsp.israelensis ondevelopment of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae). Applied and Environmental Microbiology, 63: 1095-1101.)

Method used

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  • Novel Bt protein Cry53Ab1, coding gene thereof and use
  • Novel Bt protein Cry53Ab1, coding gene thereof and use
  • Novel Bt protein Cry53Ab1, coding gene thereof and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Cloning of cry53Ab1 gene

[0027] The present invention is a new strain of Bacillus thuringiensis isolated from the soil in the virgin forest area of ​​Muchuan, Sichuan Province. The strain has been in the General Microbiology Center of the China Microbial Culture Collection and Management Committee on October 21, 2008 (Address: Beijing No. 3, Datun Road, Chaoyang District, China, deposited by Institute of Microbiology, Chinese Academy of Sciences, 100101), classified and named as Bacillus thuringiensis, and deposited as CGMCC No.2719.

[0028] In this example, the full-length sequence of cry53Ab1 gene was cloned by the following method.

[0029] The total DNA of the strain BtMC28 was extracted using a genomic DNA purification kit (purchased from Cybersun). Design primer sequence as follows:

[0030] P1: 5’ATGAATTCATATCAAAATAAAAATG 3’

[0031] P2: 5’TTATTCGACAAATAAACTATTTACA 3’

[0032] PCR reaction system:

[0033] 10×buffer 2.5μl

[0034] MgCl 2 (25mM) 1.5μl

[0035] Ta...

Embodiment 2

[0041] Example 2 Expression of cry53Ab1 gene and determination of insecticidal activity

[0042] According to the sequences at both ends of the open reading frame of cry53Ab1 gene, a pair of specific primers cry5 3F: 5′-GCG was designed and synthesized CATATG (NdeI)ATGAATTCATATCAAAATAAAAATG-3, cry5 3R: 5′-CG GAATTC (EcoR I)TTATTCGACAAATAAACTATTTACA-3', primers Nde I and EcoR I restriction sites at the 5'end respectively. Using BtMC28 total DNA as template for amplification, the reaction procedure and reaction system were the same as in Example 1. The amplified product was double digested with Nde I and EcoR I, and the digested product was the same as the vector pET-30a after double digestion. (+) After connecting, transforming E.coli DH5α competent cells, extracting their plasmids, enzyme digestion electrophoresis, and verifying that the inserted fragment size meets the expected purpose ( figure 2 ), and then transferred to the recipient bacteria E.coli.BL21(DE3). The recombi...

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Abstract

The invention provides new Bt protein Cry 53Abl and encoding genes thereof. The protein has the amino acid sequences shown in the SEQ ID NO.2 or the protein has the amino acid sequences shown in the SEQ ID NO.2 which are substituted, lacked and / or added with one or a plurality of amino acids and have the same activity. The protein can be used for preparing Bt pesticide. The gene can be transformed into crops such as cotton, corn, vegetables and the like which are caused to have corresponding anti-insect activity, thus reducing the usage volume of the pesticide, reducing environmental pollution and having important economic value and application prospect.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a new Bt protein and its coding gene and application. Background technique [0002] In the process of human production, insect pests are an important factor that causes agricultural production losses and affects human health. According to FAO statistics, the annual economic loss caused by insect pests in agricultural production worldwide is as high as 14%, disease loss is 12%, and weed loss is 11 %. The loss amounted to US$126 billion, equivalent to half of China's total agricultural output value and more than four times that of the UK. In addition, mosquito-borne diseases occupies an important position in preventive medicine, among which mosquito-borne diseases such as dengue fever and yellow fever have strong transmission power, wide spread, high incidence, and great harm. According to WHO statistics, there are as many as 80 million people infected with dengue fever in the world each ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/325C12N15/32C12N15/63C12N5/10C12N15/82A01N63/02A01P7/04
CPCY02A40/146
Inventor 李平郑爱萍朱军谭芙蓉王玲霞王世全邓其明李双成刘怀年宋福平束长龙张杰
Owner SICHUAN AGRI UNIV