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Studies on the Functions of crylAc Gene from Bacillus Thuringiensis by Error-prone PCR

Author: ZhuZhou
Tutor: ZhangYouMing
School: Hunan Normal University
Course: Microbiology
Keywords: Error-prone PCR Bacillus thuringiensis Cry1Ac gene
CLC: S476.1
Type: Master's thesis
Year: 2011
Downloads: 75
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Abstract

Bacillus thuringiensis (Bacillus thuringiensis, referred to as Bt) is the world's most widely used insecticide microorganisms, its insecticidal activity comes mainly from the sporulation insecticidal proteins produced during the research for the study of the insecticidal protein structure and function It is currently a hot topic. Bacillus thuringiensis Cry1Ac protein expression for the present is the most toxic to lepidopteran insects, and one of the most studied Bt protein, not only in agricultural production, forest management, mosquito control and water environment ecosystem more widely used, but the Cry1Ac protein structure and function of the correlation is not entirely clear, so it is necessary to use the gene mutation to a more in-depth explanation. This paper uses error-prone PCR method the Bacillus thuringiensis crylAc gene mutations screened from the mutant strains and bollworm insecticide virulence improve mutant protein, resulting in changes in the physical and chemical properties of the Cry1Ac protein before and after the mutation The structure and function of bioinformatics evidence. We chose a different Mg2 and Mn2 concentration of error-prone PCR experiments, Cry1Ac gene was amplified through the mutant gene sequencing optimum PCR conditions. After that, connect it to with Bt promoter and terminator pHT315 plasmid construct recombinant plasmid pHT315ptAc. Hit the plasmid heat transformed into E. coli Top10 competent we use the the fast pyrolysis identified transformants. We then pick a plurality of positive transformants was inoculated into the same medium, to extract plasmid electrically Go competent cells of the Cry-B. The identification of mutant strains of screening, rescreening, got some mutant. The mutant bioassay, and statistical analysis of the results obtained by the mutant 2-23, the mutant strain of cotton bollworm insecticide virulence than the wild-type strain has been significantly improved. Microscopy and SDS-PAGE analysis of mutant 2-23 Cry1Ac protein, compared with the wild-type strain differences are not significant in the microscopic morphology and SDS-PAGE on the test results. But their bioassay results indicate that the mutant 2-23 protein than the wild-type strain virulence of H. armigera significantly improved. Cry1Ac mutants 2-23 497 gene mutations, found in the extract the mutant 2-23 pHT315ptAc plasmid and DNA sequencing of the Cry1Ac protein of 166 amino acid asparagine by serine. Analysis showed that the locus of the mutation resulted in an increase alpha5 spiral overall hydrophobicity, alpha5 spiral binding with the receptor is also conducive to combining with the hydrophobic lipid interface, which improves the efficiency and capacity of the ion channel-forming. Also found that mutation of serine alpha7 helix 250 tyrosine hydrogen bonds connected to students break through the analysis of the three-dimensional structure. Breaking of hydrogen bonds may cause the viscosity of alpha 5 and alpha7 spiral of decline, the Cry1Ac protein binding to the receptor, alpha 5 and alpha7 spiral, more prone to conformational changes, resulting in α4-loop-α5 hairpin structure is easier to form. The above points may be reasons for the improvement of the mutants generated 2-23 Cry1Ac protein virulence.

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