Transformation Of Bt Chitinase Gene Chi7 Into B.subtilis

A new chitinase gene, designated chi7 (GenBank accession number AY074882), from Bacillus thuringiensis (abbr. Bt) strain Bt50 with high chitinase-producing ability was cloned into Escherichia coli JM109 with pMD-18T cloning vector. The promoter of rpsD, designated rpsP, from Bacillus subtilis subsp. subtilis strain 168 (GenBank accession number NC₀00964) was cloned by PCR. Then chi7 and rpsP were ligated with shuttle vector pAD123 and cloned into JM109 to yield the recombinant plasmid pAD-rpsP-chi7.The nucleotide sequences of chi7 and rpsP shared minxium 97.0% identity with known chitinase genes and promoter of rpsD, respectively. Especially, the rpsP sequence was only one-base difference. The recombinant plasmid pAD-rpsP-chi7 was transferred into Bacillus subtilis subsp. endophyticus strain BS-2 by electroporation and the transformants were verified by PCR and restriction analysis. The genetic stability and the biologic control of BS-2 engineering strain were preliminary analysed. The rate of plasmid loss was higher in solid culture than that in liquid culture. The inhibiting activity to Rhizoctonia solani and Botrytis cinerea of BS-2 engineering strain was much more efficient than that of BS-2 strain.