The First Cry2Ac-Type Protein Toxic to Helicoverpa armigera: Cloning and Overexpression of Cry2ac7 Gene from SBS-BT1 Strain of Bacillus thuringiensis.

The Cry (crystal) proteins from are known to have toxicity against a variety of insects and have been exploited to control insect pests through transgenic plants and biopesticides. SBS BT-1 carrying the genes was isolated from soil samples in Pakistan. The 2-kb full length gene was cloned, sequenced, and submitted to the EMBL DNA database (Accession No. AM292031). For expression analysis, DH5α was transformed with the fragment sub-cloned in pET22b expression vector using I and dIII restriction sites, and later confirmed by restriction endonuclease analysis. To assess the toxicity of Cry2Ac7 protein against lepidopteran and dipteran insects, BL21 (codon plus) strain of was further transformed with the recombinant plasmid. The 65-kDa protein was expressed in the form of inclusion bodies up to 180 OD units per liter of the medium. Inclusions were washed with a buffer containing 1.5% Triton-X 100 and >90% pure Cry2Ac7 was obtained. The inclusion bodies were dissolved in 50 mM K₂CO₃ (pH 11.5), dialyzed, and freeze-dried. This freeze-dried protein as well as inclusion bodies were used in bioassays against larvae of and . The freeze-dried protein was toxic to larvae with an LC value of 131 ng/mL. However, Cry2Ac7 produced in did not show any mortality to larvae. This is the first report of Cry2Ac protein toxic to .