Abstract

The Insecticidal protein (ICP) gene from Bacillus thuringiensis var, kurstaki HD-1 was cloned in pBluescript SK(+) vector and charicterized by overexpression in Escherichia coli XL1-blue. Total plasmids in the B. thuringiensis were isolated and digested with restriction enzyme BamHI. Then, southern blot was performed with a probe to locate the gene in the fragments. The hybridized 3.8 kb NdeI DNA fragment was cloned into the SmaI site of pBluescript SK(+) and named pHLN1-80 in forward orientation to the lacZ gene promoter and pHLN2-80 in reverse orientation to the lacZ gene promoter. Determination of 153 bp nucleotide sequence of 5'-end of the NdeI fragment in the pHLN1-80 clone revealed that there are-80 bp region of the ICP gene promoter and +73 bp region of the ICP gene at the 5'end of the ICP gene. In addition, the-80 bp promoter of the ICP gene contained transcription initiation point G at-77 bp point and BtI promoter and Shine-Dalgarno sequence at-14 to-4 bp region. The two clones showed strong insecticidal activity against 3rd the instar Bompyx mori larvae. SDS-PAGE analysis revealed that the pHLN2-80 clone clearly produces distinguishable amount (27 times more) of the 130 kDa ICP band and 100 times the insecticidal activity than that of the clone pHLN1-80. These marked differences in production and toxicity due to different orientations of the gene in the vectior provide us valuable points for further study on the ICP gene transcription at the molecular level.

• Keywords: B. thuringiensis; δ-endotoxin and gene; overexpression; insecticidal protein gene