[Characterization and cloning of the cry2A gene from indigenous isolates of Bacillus thuringiensis]

Discovery of novel cry genes of Bacillus thuringiensis (Bt) with higher toxicity is important for the development of transgenic Bt crops resistant to target pests. Two new indigenous isolates of Bt were characterized for their colony type, crystal inclusion and toxicity with Helicoverpa armigera Hubner and Spodoptera litura Linn. Screening of cry2A genes from the two new isolates of Bt, T30 and T48, by PCR with cry2A family primers showed the presence of cry2A genes only in the isolate T30. Further screening of these isolates of Bt with three different cry2A gene specific primers showed the presence of cry2Aa, cry2Ab and cry2Ac genes in both of the new Bt isolates. The cry2A open reading frame (ORF) of Bt T48 (~1.9 kb) was amplified and cloned in a T/A vector (~2.8 kb). All the E. coli transformants showed only the cry2Aa gene. Comparison of the nucleotide sequence data generated from the cry2Aa (~1.9 kb) gene showed a 99 percent homology and one amino acid variation when compared with the holotype sequence of Cry2Aa1.