Cloning, Expression Of Gene Encoding Bacillus Thuringiensis Chitinase And Construction Of Engineering Bacteria

The chitinase gene named chi7(2025bp) from Bacillus thuringiensis strain WB7 with high chitinase-producing ability was cloned in Escherichia coli DH5 a with pMD-18T cloning vector by the PCR method. The GenBank accession number ofchil was AY074882. With the software of Blast online, the sequence analysis showed that more than 90% high degree of identity with other BT chitinase gene and Bacillus cereus chitinase gene; moreover, it showed lower identity with other Bacillus chitinases gene, such as B. circulans and B. subtilis, 52% and 50% respectively. The amino acid sequence of Chi7 chitinase was also deduced. The functional and spacial structure was analysized. With the help of SMART software online for functional analysis, the sequence of 674 amino acids includes a signal peptide(7-26), a ChiA chitin catalytic domain, a fibronectin-like domain (485-557), and a chitin-binding domain(579-672). With the signal peptide analysis software TMHMM2.0, the preceding peptide of Chi7 was a transmembrane peptide, the raw cleavage site of Chi7 chitinase was between Ala-Asp at 32-33 with the SignalP analysis. With the help of ExPASy Proteomics tools, the spacial structure analysis showed that it may exist a coiled coil region at amino acid residue 200-300; there was no leucine zipper structure in the whole amino acid sequence; with PredictProtein software online of Colombia University for structure analysis, it formed 19.56% helix, 25.78% sheet, and 54.67% loop in secondary structure of Chi7 chitinase; the whole Chi7 protein appeared as compact, as a globular domain in tertiary structure. An expression vectorpBluescript SK was constructed and detected BT chitinase activity was detected in E. coli, but the chitinase activity was not high. The Bt chil gene was ligated with the plasmid pUS186, a kind of Bacillus subtilis expression plasmid, then transformed to B. subtilis strain QB1098 by electroporation. The recombinant plasmid pUS-chil was also ' transformed to B. subtilis strain BS-2, detected by PCR method. Therefore, 5 engineering bacteria strains of B. subtilis with BT chitinase gene were obtained and kept.