[Cloning and expression of ICP cry1AB16 gene of Bacillus thuringiensis]

With two pairs of primers designed on the basis of the sequence of cry1 and cry1Ab gene from Bacillus thuringiensis (Bt), a novel insecticidal crystal protein crylAb gene of Bt was amplified from bacillus strain AC-11 by using Long Template PCR System. Southernblot further confirmed that the novel gene existed in plasmid of the strain. The amplified fragment was sequenced and compared with cry1Ab1 gene in EMBL/GenBank. The results showed that eight nucleotides and seven amino acid residues were different from that of cry1Ab1, suggesting that it was a new crylAb gene, which was designated cry1Ab16 in GenBank (Accession No. AF375608). The cry1Ab16 gene was cloned into the E. coli expression vector pQE30, creating the recombinant plasmid pQCT, which was then transformed into E. coli M15. Westernblot analysis showed that Cry1Ab16 protein, induced by IPTG was expressed in the strain M15 (pQCT) with the molecular mass of approximate 130 kD, but Cry1Ab16 was unstable and was mostly degraded into about 65 kD protein. Bioassay showed that the LC50 of Cry1Ab16 against the third instar lavae of Plutella xylostella with a spreaded method was 258.3 mg/L, and it could also inhibit the growth of Spodoptera larvae.