Thirty nine Bt Cry1 subgroup protein sequences were retrieved from NCBI and analyzed for physicochemical properties, active site and relationship in relation to their variations in toxicity. Cry1 proteins were found to be hydrophilic and stable. SOSUI server predicted presence of two transmembrane regions in Ag and a single transmembrane region from Aa to Ae. EMBOSS PepWheel tool analysis of the transmembrane regions showed that there were 23 highly conserved residues towards the N terminal which are hydrophobic and more than half of the residues were neutrally charged. No signal peptide was detected which classifies the Cry1 group proteins as non-secretory proteins. Cry1 proteins have very high composition of neutral amino acids and might transform into negative charge after solubilization in alkaline environment (insect midgut). The negatively charged protein might misfold causing difficultly to digest and thereby be toxic to lepidopteran. Active sites of Cry1 proteins with more than 50% neutral amino acids showed wide insecticidal spectrum and further positive correlation (r = 0.7731) was observed between neutral amino acids and insect species affected (Y = -138.21 + 2.907X). Similarity of sequences was found between Cry1 proteins based on their high or low spectrum of insecticidal activity.
Bacillus thuringiensis (Bt) strains were isolated from jhum-agriculture, jhum-forest, aquatic and fallow soil samples from Mizoram by acetate selection method. Isolates were characterized for biochemical typing, cry gene and protein profiling, growth curve study and toxicity against Culex tritaeniorhynchus. Bt frequency was high in jhum-agriculture land (69.56%) whereas low in jhum-forest soils (31.57%). Bt was found to be abundant in jhum shifting cultivation soil with an index ranging between 0.010 and 0.015. Majority of the isolates from jhum soils produced oval and spherical crystals and showed eleven types of crystal proteins groups. PCR analysis revealed predominance of dipteran-active cry genes (cry4 and cry9). The variations in crystal morphology, cry genes and Cry protein (s) from the isolates of Bt revealed molecular diversity. Higher mortality, lower lethal dose, and lesser time to kill were observed in Bt isolates from jhum soils than aquatic and fallow habitats. Based on the toxicity test, SC1 and HP7 isolates containing cry 4 and cry 9 genes showed higher activity. Growth curve analysis showed significant variations among Bt isolates to reach the sporulating stage. Higher growth index and lower mean generation time were observed in SC1 and HP7 Bt isolates. Bt strains express different endotoxin genes and crystal proteins and their harvesting time also varied from strain to strain. Significant variation was found in Bt isolates from jhum habitats in relation to the cry gene composition, protein profiling and toxicity. Results from this study suggest that novel Bt entomopathogens may complement for regulating mosquito vectors.
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