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20131
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Subject: Agricultural Biotechnology × Author: A.S. Pooja ×
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    ThesisItemOpen Access
    Profile of cry from Native Bacillus thuringiensis Isolates and Expression of cry1l
    (UAS, Dharwad, 2013) A.S. Pooja; S.K.Prashanthi
    The characterization of 255 Bacillus thuringiensis isolates of Coorg, Sharavati and BR Hills containing genes known to be active against coleopterans viz., cry1I, cry3, cry7, cry8, cry14, cry18, cry26, cry28, cry34, cry35, cry36, cry23, cry55, cry37 and lepidopteran specific genes like cry1, cry2, cry8, cry9, cry20, cry1Aa1, cry1Ab2, cry1Ac1, cry1Ad1, cry1Ae1, cry1Ca1, cry1Da1, cry1Ea1, cry1Fa1, cry1Ia1, cry2Aa1, cry2Ab1, cry2Ac1, cry9Aa1, cry9Ca1 was done through PCR amplification using the specific and degenerate primers. The isolates were also tested for their insecticidal activity against P. xylostella. Among the coleopteran specific genes, the most predominant was cry1I gene, present in 18 isolates at a frequency of 7.05 per cent. cry1 gene was most abundant (35.39%) followed by cry2 (33.62%) among lepidopteran specific genes. A variant of cry1I gene based on amplicon restriction fragment length polymorphism (ARFLP) was cloned into pTZ57R/T, transformed into E. coli DH5a and sub-cloned in an expression vector pQE-30 after amplification of a 2169 bp DNA fragment of cry1I gene from Bacillus thuringiensis isolate DBT189, the sequence of which showed 99 per cent homology with known cry1Ia gene from Bacillus thuringiensis subsp. kurstaki suggesting that there is one per cent sequence variation with respect to reference strain and 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 SDS-PAGE analysis indicating the expression of cry1I gene.