Profile of cry from native Bacillus thuringiensis isolates and expression of cry1I
AbstractThe characterization of 255 Bacillus thuringiensis isolates of Coorg, Sharavatti and BR hills, containing genes known to be active against coleopteran and lepidopteran insect species was done through PCR amplification using the specific and degenerate primers. The isolates were also tested for their insecticidal activity against Plutella xylostella. Among the coleopteran specific genes screened, the most predominant was cry1I gene which was present in 18 isolates at a frequency of 7.05%. cry1 gene was found to be most abundant (35.39%) among the lepidopteran specific genes. A variant of cry1I gene based on amplicon restriction fragment length polymorphism (ARFLP) was cloned into pTZ57R/T and subcloned in an expression vector pQE-30 after amplification of a 2169 bp DNA fragment of cry1I gene from B. thuringiensis DBT189, the sequence which showed 99% homology with known cry1Ia gene from B. thuringiensis subsp. kurstaki. There were six mismatches between the two amino acid sequences. The cry1I- type gene consisted of an open reading frame of 2124 bp that would encode for 720 amino acids. An expected band size of 81 kDa was observed after sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicating the expression of cry1I gene.
Keywords: Bacillus thuringiensis, Plutella xylostella, ARFLP, cloning, SDS-PAGE
African Journal of Biotechnology Vol. 12(22), pp. 3545-3562