CLONING AND EXPRESSION OF crylA(c) GENE FROM NATIVE Bacillus thuringiensis (Berliner) ISOLATES
Loading...
Date
2003
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Agricultural Science, Dharwad
Abstract
The focus of the present study was to clone the full length and truncated CrylA(c)
gene from native Bacillus thuringiensis isolates. Bioassay of certain native isolates of
B. thuringiensis along with reference strain HD-1 showed that a native isolate, D-1
showed the maximum mortality of 90.6 per cent at 72 hours. Among the 21 isolates
tested for presence of CrylK gene D-1, PP-6, PP-9 possessed CrylA (a), CrylA (b),
CrylAic). PP-7, SI-3, D-21 did not possess any of these genes.
Primers were designed to pickup full-length 3.5Kb Crv'IA(c) and truncated
2Kb Cry IA(c) gene, using the conserved regions of Cry gene. The amplified products
using these p rimers were c loned into pTZ57R at EcoZll s ite containing T- o verhangs
through T/A cloning strategy. Of the several clones obtained, two clones pKM1308 and
pKM1508 harbouring 3.5Kb and 2Kb respectively were analysed further. The presence
of insert in the constructs has been confirmed by restriction analysis, PCR amplication
and plasmid preparation. Restriction of pKM1308 recombinant clone with BamUl
resulted in production of two fragments one of 3500bp corresponding to full length
CrvlA(c) and another 2868bp corresponding to vector. The restriction of 2.0kb
recombinant with BamUl yielded a single band of 4868 bp indicating the presence of the
insert. The recombinants pKM1308 and pKM1508 were sequenced. The obtained
sequences v/ere used for nucleotide-nucleotide BLASTn search to know homology with
different B thuringiensis genes. The gene fragments showed 98% homology to many of
the Cry genes including c/7lA(c), crylA{a), cryIA(b) and cryllS . Blast search also
revealed that the 3.5Kb was cloned in reverse orientation in pKM1308 and the 2.0 Kb
insert was in correct orientation in pKM1508.
Description
Keywords
null
Citation
No. of references 90