Bt protein Cry52Bal as well as encoding gene thereof and application thereof

A cry52ba1, bt protein technology, applied in the new Bt protein and its coding gene and application field

Active Publication Date: 2009-09-16
SICHUAN AGRI UNIV
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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 M C, 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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  • Bt protein Cry52Bal as well as encoding gene thereof and application thereof
  • Bt protein Cry52Bal as well as encoding gene thereof and application thereof
  • Bt protein Cry52Bal as well as encoding gene thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1c

[0025] Cloning of embodiment 1cry52Bal gene

[0026] The present invention is isolated from the new strain of Bacillus thuringiensis (Bacillus thuringiensis) obtained from the soil in the virgin forest area of ​​Muchuan, Sichuan Province. No. 3, Datun Road, Chaoyang District, City, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101) is preserved, and the classification is named Bacillus thuringiensis (Bacillus thuringiensis), and the preservation number is CGMCC No.2871.

[0027] In this example, the full-length sequence of the cry52Bal gene was cloned by the following method.

[0028] Total DNA of BM59-2. The primer sequences were designed as follows:

[0029] P1: 5'ATGAATTCATATCAAAATAAAAAATG3'

[0030] P2: 5'TTACCTCCTACTTCAGTACATACTTT3'

[0031] PCR reaction system:

[0032] 10×buffer 2.5μl

[0033] MgCl 2(25mM) 1.5μl

[0034] Taq enzyme 0.2μl

[0035] dNTPs (2.5mM) 2μl

[0036] Primer 2μl

[0037] Template 5μl

[0038] Final reaction volume ...

Embodiment 2

[0040] Example 2 Expression of cry52Bal gene and determination of insecticidal activity

[0041] According to the sequence at both ends of the open reading frame of the cry52Bal gene, a pair of specific primers cry53F: 5′-GCG was designed and synthesized CATATG (NdeI)ATGAATTCATATCAAAATAAAAATG-3, cry53R: 5'-CG GAATTC (EcoR I) TTACCTCCTACTTCAGTACATACTTT-3', respectively at the 5' end primers Nde I and EcoR I restriction sites. The BM59-2 plasmid was used as a template to amplify, and the amplified product was double-digested with Nde I and EcoR I, and the digested product was ligated with the vector pET-30a(+) after the same double-digestion, and transformed into E.coli After DH5α competent cells were extracted and its plasmid was digested and electrophoresis verified that the size of the inserted fragment met the expected purpose ( figure 2 ), and then transferred into the recipient strain E.coli.BL21(DE3). The recombinant plasmid was named pET-52Ba, and the recombinant c...

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Abstract

The invention provides a new Bt protein Cry52Bal as well as an encoding gene thereof; the protein is provided with an aminoacid sequence as shown by SEQ ID No.2 or the protein which has the same activity and is obtained by substituting, deleting and / or adding one or plural aminoacid to an amino acid sequence as shown by SEQ ID No.2. The protein of the invention can be used for preparing Bt insecticides, the gene can be used for converting cotton, maize, rice, vegetables and other crops to ensure that the crops have corresponding anti-insect activity, thereby reducing the use of agricultural chemical, alleviating environment pollution, and having significant economic value and application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel 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 losses caused by insect pests in agricultural production worldwide are as high as 14%, disease losses reach 12%, and weed damage losses reach 11%. %. The loss was as high as 126 billion US dollars, equivalent to half of China's total agricultural output value, and more than four times that of the United Kingdom. In addition, mosquito-borne diseases occupy an important position in preventive medicine, among which mosquito-borne diseases such as dengue fever and yellow fever have strong transmissibility, wide prevalence, high incidence and great harm. According to WHO statistics, as many as 80 million people are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/325C12N15/32C12N15/63C12N5/10A01N63/02A01P7/04C12N15/82C12R1/07A01N63/50
CPCA01N63/02C07K14/325C12N15/8286Y02A40/146A01N63/50A01N63/23
Inventor 郑爱萍李平朱军王玲霞王世全邓其明李双成刘怀年
Owner SICHUAN AGRI UNIV
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