Cloning and Characterization of Cry1 and Cry2 Genes from Indigenous Bacillus thuringiensis Isolates

Abstract

Insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) are toxic to specific insect pests. They are used as alternatives to chemicals for controlling insect pests using transgenic plants and biopesticides. Three strains of Bacillus thuringiensis expressing cry1 and cry2 genes were identified in soil samples collected from different locations in India. The full-length cry1, cry1I, and cry2 type genes (3.5, 2.1, and 1.9 kb, respectively) were cloned and sequenced. The full-length clones of the three genes were sub-cloned in the E. coli expression vector, and expressed proteins were analysed. The expression of cry1, cry1I, and cry2 resulted in 132, 81, and 70 kDa proteins, respectively. The expressed proteins were tested against secondinstar larvae of Helicoverpa armigera. The expressed proteins of cry1 and cry2- type from BRI-86 exhibited the highest toxicity to H. armigera larvae with an LC50 of 2.34 and 0.2 µg/ml diet, respectively. The LC50 exhibited by the test proteins was two and four-fold lower than the reference Btk HD-1 proteins. Thus, the spore preparation or the genes can be used individually or in combination to control insect pests.