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Novel disinsection Bt protein Cry54Aa1, coding gene thereof and use

A bt protein, gene technology, applied in applications, pesticides, genetic engineering, etc.

Inactive Publication Date: 2009-08-05
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 disinsection Bt protein Cry54Aa1, coding gene thereof and use
  • Novel disinsection Bt protein Cry54Aa1, coding gene thereof and use
  • Novel disinsection Bt protein Cry54Aa1, coding gene thereof and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Cloning of cry54Aa1 gene

[0026] The present invention is a new bacterial strain of Bacillus thuringiensis (Bacillus thuringiensis) isolated 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.2719.

[0027] In this example, the full-length sequence of the cry54Aa1 gene was cloned by the following method.

[0028] The total DNA of strain BtMC28 was extracted using a genomic DNA purification kit (purchased from Saibaisheng Company). The primer sequences were designed as follows:

[0029] P1: 5'ATGAGTATGAAATCATTGATTC3'

[0030] P2: 5'CACGTCAGGGGTAAATTCGATT3'

[0031] PCR reaction system:

[0032]10×buffer 2.5μl

[0033] MgCl 2 (25mM) 1.5μl

[0034] Taq enzyme 0.2μl

[0035] dNTPs (2.5m...

Embodiment 2

[0040] Example 2 Expression of cry54Aa1 gene and determination of insecticidal activity

[0041] According to the sequence at both ends of the open reading frame of the cry54Aa1 gene, a pair of specific primers cry54A: 5′-GCG was designed and synthesized CATATG (NdeI)ATGAGTATGAAATCATTGATTC-3'; cry30R: 5'-CG GAATTC (EcoR I) CACGTCAGGGGTAAATTCGATT-3', respectively at the 5' end primers Nde I and EcoR I restriction sites. The total DNA of BtMC28 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.coliDH5α sensory state cells, extract their plasmids and electrophoresis to verify that the size of the inserted fragment meets the expected purpose ( figure 2 ), and then transferred into the recipient strain E.coli.BL21(DE3). The recombinant plasmid was named pET-54Aa, and the recombinant containing the recombi...

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Abstract

The invention provides a new Bt protein Cry54Aa1 and an encoding gene thereof. The protein has an amino acid sequence shown in an SEQ ID No.2 or has coordination activity after replacing, deleting and / or increasing one or more amino acids in the amino acid sequence shown in the SEQ ID No.2. The protein can be used for preparing the Bt pesticide, and the gene transformation can be performed to agricultural crops such as cotton, corn, rice, vegetables, and the like so that the agricultural crops have the corresponding binding activities, therefore, the consumption of the pesticide and the environmental pollution are reduced, and the invention has important 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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IPC IPC(8): C07K14/325C12N15/32C12N15/82C12N5/10A01N63/02A01P7/04C12R1/07
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
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