Screening of new isolates of Bacillus thuringiensis (Bt) and cloning of the cry genes

Abstract

Nine new indigenous isolates of Bacillus thuringiensis (Bt) were characterized for their colony type, crystal inclusion and toxicity analysis with Helicoverpa armigera Hubner and Spodoptera litura Linn. Genomic deoxyribonucleic acid (DNA) isolated from all the new isolates were subjected to screening for cry1, cry2, cry4, cry10 and cry11 genes and predicted possible potential DNA amplicons were cloned and sequenced. Partial cry1 gene fragment (~1.5 kb) amplified by degenerate primers and about 450 bp DNA fragment amplified by cry10 gene specific primers from two isolates T109 and T136 were cloned in to T/A cloning vector. DNA sequencing of about 1.5 kb amplicon showed 99% homology to the holotype sequence of cry1Ac1. Nucleotide sequence of about 450 bp fragments of isolate T109 and T136 showed homology to a hypothetical protein and serine/threonine phosphatase respectively.

Keywords: Bacillus thuringiensis (Bt), cloning, cry genes, polymerase chain reaction (PCR), toxicity analysis