Study on Effect of Transcription Factor CodY Protein in Bacillus Thuringiensis
|School||Huazhong Agricultural University|
|Keywords||Bacillus thuringiensis Transcription Factor CodY Parasporal crystal protein|
Bacillus thuringiensis is a capable of producing Bacillus soil bacteria form spores as well as a large number of parasporal crystal form of insecticidal crystal protein in the stabilization period. Crystal protein metabolism regulation mechanism the Bacillus thuringiensis one of the hot, spore formation mechanism has been a breakthrough, crystal protein metabolic regulation mechanism still do not have a reasonable and clear explanations. CodY is a global transcriptional regulator in Gram-positive bacteria, can regulate the expression of over 200 genes, most of them is the stability of expression of genes, including many virulence genes of pathogenic microorganisms. The current study reported on CodY thuringiensis Bacillus subtilis in function rarely, this study mainly by building over-expression Cody protein of Bacillus gold engineering Bacillus bacteria and knock addition to cody gene mutation strains to analyze the role of mechanisms of the protein in Bacillus thuringiensis trying to find Cody protein and parasporal crystal protein formation and metabolic regulation, and thus lay the foundation for building the entire Bacillus thuringiensis metabolic regulatory networks. Our laboratory has published thuringiensis gold Bacillus subtilis BMB171 and CT-43 and not fully released YBT-1520 of genome-wide set of sequences, Cody protein bioinformatics analysis, results show that it is a wax-like Bacillus subtilis The nucleotide similarity of more than 99%, but with a Bacillus subtilis compare the difference is relatively large, and in cody genes, chromosomes on the downstream gene of Bacillus subtilis is also very obvious difference compared. The experimental use of an expression vector pHT1K-Plip-TS, to build overexpression Cody protein the recombinant vector pHT1K-PlipcodyTS. The recombinant vector was transformed into the YBT-881, Bacillus thuringiensis wild-type strain, recombinant strain YBT-881-L1. After its characterization showed that: the YBT-881-L1 and wild-type strains growth curve and PARASPORAL of crystal morphology no significant difference, however, found by SDS-PAGE and mass spectrometry analysis and RT-PCR: engineering bacteria compared with the wild-type strain, two strains All Cryl class of proteins containing the same, while in the engineering strain silence cry2Ac gene is activated expression, and produce large amounts of Cry2Ac protein sequence alignment after the to confirmed the cry2Ac gene cry2Ac4 similar rate of 99%, this makes engineered bacteria containing the Class Cry2A Cryl two crystals protein. As for the stimulation Cry2Ac protein expression reason needs further study. In this study, a temperature-sensitive carrier built knockout vector pBMB0413, to construct the cody gene deletion mutant by homologous recombination in Bacillus thuringiensis BMB171. Amplified by PCR, Western blot analysis and RT-PCR validation can not transcriptional expression of the mutant Cody protein. In this mutant characteristics identified that: mutant can not form a complete Bacillus normal. And mutant strains PHB content reached the highest value after 12h, during the stable period is not degraded but maintained at a stable level, while in PHB BMB171 content declined sharply after 8h to 10h reaches the highest value, and analysis of the reasons: PHB is a carbon source hoards of cell growth to stationary phase, due to a lack of nutrition, the need to consume the use of PHB bacteria produce spores also need a carbon source and energy substances. Thus, PHB will sharply. Mutant is not capable of forming a normal Bacillus PHB will not be degraded and used.