Site-directed mutagenesis in Cry proteins of Bacillus thuringiensis to demonstrate the role of domain II and domain III in toxicity enhancement toward Spodoptera litura

Abstract Background Bacillus thuringiensis ( Bt ) is a gram-positive bacterium responsible for the production of wide variety insecticidal Cry, Cyt, and Vip proteins with distinct insect specificities. The bioinsecticides derived from Cry account > 95% microbial biopesticide market to combat pest species belonging order Lepidoptera (including Spodoptera spp.), Coleoptera, Diptera, etc. are engineered by using different molecular techniques control development multiple insecticide resistance problems in major pests bio-toxicity assays. Main body It common knowledge that domain II III involved pore formation or interaction between several larval receptors membranes epithelial cells. In present research, PCR site-directed mutagenesis technique was used introduce total four mutations into cry genes 1 2) near key regions active proteins. diet overlay bioassay test efficacy expressed mutant against litura (Fabricius) (Lepidoptera: Noctuidae), one most damaging bollworms cotton, causing severe output losses. Results Two amino acid replacements receptor binding Cry1Ac toxins (S573, L588) showed decrease activity mutants. However, substitutions binding/pore Cry2Aa (T325, S445) were some extent proved more toxic than wild-type recombinant protein, an increase mortality percentage 3.33 6.66% after 24 72 h treatment, respectively, 2nd instar larvae S. . Conclusion A comparison demonstrated susceptible those being treated toxins. LC 50 values mutants slightly reduced