Bacillus thuringiensis subsp. galleriae HD29 (Bacillus Genetic Stock Center ID 4G5; Serotype 5a5b) is known to harbor multiple Cry-type genes such as cry1Cb, cry1Db, cry1Ga, cry2Ab, cry2Ac, cry9A and cry9B, which produce crystals consisting of 65- and 130-kDa proteins as major constituents. A new cry2Ad gene coding for entomocidal crystal protein is being reported. The main objectives were (i) to clone, sequence and express the new cry2Ad gene coding for entomocidal crystal protein of Bacillus thuringiensis subsp. galleriae HD29 (ii) to determine the toxicity of this new toxin to insect pests and (iii) to determine and characterize the salient features of 3D structure of the protein. Routine methodology was adopted for cloning and sequencing of the gene. For expression analysis, cry2Ad5 gene was sub-cloned in pET22b expression vector and transferred to E. coli BL21 (codon plus) for expression. Proteins expression was analyzed on 12% SDS-PAGE. The expressed protein was used for determining insecticidal activity against lepidopteran (Helicoverpa armigera, Spodoptera litura) and dipteran (Aedes aegypti, Culex quinquefasciatus) insects. cry2Ad5 gene was cloned and sequenced (EMBL accession number AM765844). The protein, listed as Cry2Ad5 (https://www.bpprc-db.org/search_database), was expressed in the form of inclusion bodies, was found to be more toxic to A. aegypti than C. quinquefasciatus larvae. No significant effect was detected against Helicoverpa armigera. The deduced amino acid sequence with 633 amino acid residues shows only 96% identical residues when compared with other δ-endotoxins of the Cry2 family. The predicted 3-D model of Cry2Ad5, when compared with other Cry2Ad type proteins, revealed two semi-conserved and one non-conserved substitution in domain I, and only one semi-conserved substitution was found in the pore forming domain II. The receptor binding motif is present in the loops 1 and 2 of Domain II. A non-significant substitution existed in the interdomain region of the protein. The docking model of Cry2Ad5 was found to be forming 161 and 16 hydrogen bonds with Cadherin and APN of Helicoverpa armigera, respectively. The new Cry2Ad5 toxin cloned and expressed from Bacillus thuringiensis subsp. galleriae strain HD 29 is more toxic to Aedes aegypti larvae than to those of Culex quinquefasciatus.
Key words: cry2Ad5 gene, Helicoverpa armigera, Genotyping, Aedes aegypti and Culex quinquefasciatus entomocidal crystal protein, Bacillus thuringiensis subsp. galleriae strain HD 29