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Molecular Characterization and Insecticidal Activity of Bacillus tluringiensis Isolates, Cloning and Expression of cry, vip Gene Variants and Mutagenesis of Codon Optimized cry1 Ac

KrishiKosh

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Title Molecular Characterization and Insecticidal Activity of Bacillus tluringiensis Isolates, Cloning and Expression of cry, vip Gene Variants and Mutagenesis of Codon Optimized cry1 Ac
 
Creator Ashwini B.K.
 
Contributor P.U. Krishnaraj
 
Subject Agricultural Microbiology
 
Description The present work involved isolation of B. thuringiensis from different
ecological niches, followed by their molecular characterization, cloning and
expression of variants of cry and vip and mutagenesis of cry. From a total of 1745 soil
samples collected, 977 isolates were identified as B. thuringiensis. The most
predominant crystalline inclusion was the spherical crystals. The presence of cry/vip
genes was determined and the most abundant gene was cry10 and cry 20. Several
isolates had more than one cry gene in them. Only ten of the isolates harboured
vip3A. Four isolates showed 100 per cent mortality at 48h of exposure against third
instar larvae of Plutella xylostella. Twenty two native isolates exhibited 100 per cent
mortality at72h. The per cent mortality of third instar larvae of Crocidolomia
binotalis ranged from 0 to 100 per cent. The reference strain HD1 exhibited 96.6 per
cent mortality, whereas the isolate DBT 763 exhibited 100 per cent mortality.
The analysis of rep PCR fingerprints showed 18 to 20 bands with size ranging
from 0.2 to 5kb with ERIC2 primer alone. One of the isolates DBT1881 showed 100
per cent similarity to B. thuringiensis subsp. andalousiensis where as DBT1853
showed 100 per cent similarity to three of the B. thuringiensis serovars viz, aizawai,
toumanoffi and roskildiensi. The variant cry1 gene from DBT381 and DBT84 based
on ARLP patterns was cloned and expressed in E. coli. The nucleotide sequence
showed 99 per cent homology with the reference cry1Ac22 (EU282379.1). The vip3A
variant was cloned from 4L3 and 4C2 and was expressed in E. coli. The codon
optimized cry 1Ac from the truncated version (1.85kb) of a native cry1Ac was
subjected to random mutagenesis and one mutant RM2G was observed to possess 20
per cent more toxicity than the cry1AcM to P. xylostella.
 
Date 2016-07-25T10:57:49Z
2016-07-25T10:57:49Z
2011
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/69695
 
Format application/pdf
 
Publisher UAS Dharwad