Sequence Analysis And Safety Research Of Enterotoxin Genes In Bt

Bacillus thuringiensis (Bt) was widely used in preventing from insect pests of agriculture and forestry, which had been a kind of microbial pesticides of success since 1956 when Bt was in commercial. Its risk was related to the health of human. While Bacillus cereus (Be) was known to all that it was a kind of conditional pathogenic bacteria, which homology was closed to Bt. Enterotoxin produced by Be was one of the important factors that cause diarrhea. According to some research, enterotoxin genes also widely exist in Bt. Consequently, this study focus on the safety of enterotoxin in Bt, to lay a theoretical foundation for large-scale application.6 enterotoxin gene primers were designed to detect the presence of enterotoxin of 40 strains of Bt from different sources in our lab by polymerase chain reaction(PCR). The results showed that 30 strains of Bt contained all of the 6 genes. And the detection rate of nheC gene reached to 100%, nheA、nheB gene reached to 95%, whose detection rate ranked only second to nheC gene. The lowest detection rate was bceT gene’s, also reached to 82.5%. In addition, the detection rate of hblA gene and ent gene was 87.5%,90%, respectively. Therefore, the enterotoxin genes were proved exist in Bt extensively.The open reading frame (ORF) of 5 enterotoxin genes from BRC-LLP29 and BRC-XQ9 was cloned, contained ent、hblA、hblB、hblD、nheA. Compared the enterotoxin amino acid sequence of Bt to that of Be using NCBI BLAST software, the results indicated that the similarity between them was very high. The ent gene was 98% identical to the similar gene of Be, and the amino acid sequence homology of hblA、hblB、hblD、nheA gene was up to 99%.nheA gene from BRC-LLP29 and BRC-XQ9 was transformed to E.coli BL21 to express NheA-pET32a fusion proteins with IPTG induced. The expression activity and biotoxicity of NheA-pET32a fusion proteins and original strains was investigated by Tecra BDE VIA Kit and rat acute oral test. The results indicated that the expression activity of NheA-pET32a fusion proteins was very high, and enterotoxin expression activity of the fermentation liquor of original strains was also detected out. However, in the rat acute oral test, it didn’t show the corresponding biological toxicity, all of the experimented rats grew up normally. The rats was dissected and their gastrointestinal mucosa was smooth, the shape、lustre and proportion to the size of each viscera had no exception. No rat died in the experimental process.Connected the pMAD plasmid and the upstream and downstream fragment of picr gene, which was toxin regulatory factor, to construct a vector for plcr gene-knockout for laying the foundation of the regulation mechanism of enterotoxin in Bt.