Cloning, characterization and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain SWK1, native to Himalayan valley Kashmir

• A new cry2A gene was cloned and expressed from a promising indigenous Bacillus thuringiensis SWK1 strain.
• B. thuringiensis δ-endotoxin Nomenclature Committee designated this new cry gene as cry2Al1.
• Expressed Cry2Al1 protein showed toxicity to Spodoptera litura and Helicoverpa armigera.

Bacillus thuringiensis (Bt) is a gram positive bacterium which is effectively being used in pest management strategies as an eco-friendly bioinsecticide. In the present study a new cry2A gene was cloned from a promising indigenous B. thuringiensis SWK1 strain previously characterized for its toxicity against Spodoptera litura and Helicoverpa armigera larvae. The nucleotide sequence of the cloned cry2A gene pointed out that the open reading frame has 1902 bases encoding a polypeptide of 634 amino acid residues with a probable molecular weight of 70 kDa. Homology comparisons showed that the deduced amino acid sequence of Cry2A had a similarity of 94% compared to that of the known Cry2Aa protein in the NCBI database and this gene has been named as cry2Al1 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry2Al1 was ligated into pET 22b vector and expressed in Escherichia coli BL21 (DE3) pLysS under the control of T7 promoter induced by isopropyl-beta-d-thiogalactopyranoside (IPTG). SDS-PAGE analysis confirmed the expression of cry2Al1 as ∼65 kDa protein. Insect pest bioassays with neonate larvae of S. litura and H. armigera showed that the purified Cry2Al1 are toxic to S. litura and H. armigera with LC50 2.448 μg/ml and H. armigera with 3.374 μg/ml respectively.

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