Molecular detection and Cloning of Cry1 and Cry2 genes of Bacillus Thuringiensis Isolates From Meerut

Abstract

Bacillus ti11Lringiensis is a ubiquitous, gram-positive and spore-fonning bacterium. During sporulation, it produces intracellular crystal proteins (cry proteins), which are toxic to insects (Aronson, 2002). Because of its insecticidal activity, it has been used for nearly fifty years to control certain insect species atnong the orders Lepidoptera, Coloeptera, and Diptera (Gillet al., 1992). In this study twenty saanples from soil were collected from Meerut. Isolation of Bt was done by Sodium acetate Method. Purified isolates were compared with growth spots of reference strains obtained from ITCC (Indian Type Culture Collection), IARI (Delhi) and around fifteen samples contain Bt. DNA isolated from native strain was used as template for amplification using two universal primers specific for cty1 and c1y2 genes. The universal primer pair Unl for cry I gene and Un2 for cry2 gene amplified the expected fragtnents of --270 bp and --550 bp for seven samples when seen in electrophoresis respectively. T'he PCR an1plified DNA fragments was cloned into the plasmid vector pTZ57Rff using traditional transformation method. Cloning was confirmed by plasmid PCR and restriction digestion of plasmid isolated from transformed colonies. Sequencing and in-silico analysis was perfonned using various bioinfonnatics tools. cry1 gene amplified in this study had highest sequence homology with cry I Ac gene and that of cry2 gene with cry2Ab gene. The identification of known cry genes in the B. thuringiensis strains is i1nportant, since the specificity of action is known for many of the Cry toxins. This fact allows the possibility of selecting native strains that could be used in the control of some targets and of selecting strains with the highest activity (Rai et al., 2011).