Study On Target Genes Of Transcription Factor CodY Protein In Bacillus Thuringiensis

Bacillus thuringiensis (Bt) is a gram-positive bacterium which can form spore and insecticidal crystal proteins (ICPs) during the stationary phase. It is widespread in nature. The metabolic regulatory mechanism of spore and insecticidal crystal proteins is always the research focus in B. thuringiensis. Currently, the research of spore is very thorough. But the regulation mechanism of the insecticidal crystal proteins is still not perfect, especially in Cry2. CodY, a global transcription factor in Gram-positive bacteria can regulate about200genes involved in formation of bacteria competence, microbial metabolism, and the virulence of pathogenic microorganisms. Up to now, the research of CodY protein in B. thuringiensis is almost blank. Our previous studies had shown that, overexpression of CodY protein in B.thuringiensis YBT-881could stimulate the formation of the silenced cry2Ac4gene. So, CodY protein played an extremely important role in the formation of the insecticidal crystal proteins.This study is based on the genome-wide sequencing of B. thuringiensis YBT-1520. We found and identified CodY targets in genome-wide level. It can supply for the mechanism of the formation of insecticidal crystal proteins. Furthermore, it can lay the foundation for building B. thuringiensis metabolic regulatory networks.1. According to complete genome sequences of B. thuringiensis YBT-1520, bioinformatics analysis of CodY protein was made by NCBI blast. The results showed that the similarity of CodY in B.thuringiensis and B.cereus group was about99%. However, compared with B.subtilis, the difference was obvious. In addition, we also analyzed the position of CodY protein gene in YBT-1520.2. An in vitro experimental technique called pull down to find and indentify CodY protein targets in the whole genome has been established. First, we applied avi-tag technology to biotinylate CodY protein and digested YBT-1520genome to100-400bp DNA fragments coupled with linker. Second, we screened for target genes of CodY protein by Streptavidin-biotin system. CodY-Ap protein could be biotinylated which had been certificated by ELISA and Western blot experiments.3. By biotin pull down we obtained46putative CodY target genes. After classify, we selected five genes which may be related with forming of the crystal protein genes for further validation. First of all, we found that four DNA fragments could interact with CodY by Bacterial one-hybrid which were in upsteam and internal of genes. We got that these genes were involved in transport of amino acid, degradation of protein and synthesis of branched-chain amino acid which were associated with the formation of insecticidal crystal proteins.