CLONING AND NUCLEOTIDE-SEQUENCE OF AN INSECTICIDAL CRYSTAL PROTEIN GENE FROM BACILLUS-THURINGIENSIS DSIR732 ACTIVE AGAINST 3 SPECIES OF LEAFROLLER (LEPIDOPTERA, TORTRICIDAE)

The gene encoding the 133 kDa insecticidal crystal protein of Bacillus thuringiensis var. kurstaki isolate DSIR732, has been cloned into Escherichia coli. The complete nucleotide sequence of the coding region of the cry 732 gene, has been determined and indicated a large open reading frame (ORF) of 3534 nucleotides encoding a protein of 1178 amino acid residues, with an estimated molecular weight of 133 330 Da. In addition, the sequence of 387 nucleotides 5' and 257 nucleotides 3' of the coding region has been determined. Sequence comparisons of this cry 732 gene and its flanking regions with those of other Bt cry genes revealed that it is a member of the cry IA(c) class of Bt cry genes. Alignment of the cry 732 nucleotide sequence with that of the cry IA(c) holotype gene sequence from Bt kurstaki HD73 revealed only four nucleotide differences in the ORF, none of which resulted in amino acid substitutions, and one nucleotide difference in the region 5' to the ORF. Nucleotide sequence comparisons suggested that the cry 732 gene may have resulted from recombination events between the cry IA(c) genes of Bt kurstaki HD73 and Bt kenyae HD558-2. The cry 732 gene has been expressed in E. coli and bioassays, using extracts of E. coli producing the 133 kDa Cry732 protein, have demonstrated that the recombinant protein retains its insecticidal properties against three species of leafrollers (Lepidoptera: Tortricidae).