MOLECULAR CHARACTERIZATION OF INSECTICIDAL GENES IN NATIVE Bacillus thuringiensis ISOLATES
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Date
2003
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University of Agricultural Science, Dharwad
Abstract
The present study was conducted to assess the molecular diversity and to
characterize the cry gene profile of native Bacillus thuringiensis isolates. Nine isolates of
B. thuringiensis and three reference strains were characterized by PCR-product profiles
using random primers obtained from Operon Technologies, USA. The nine primers used
for assessing the molecular diversity among the isolates generated 169 bands, all
polymorphic (100 % polymorphism). Among the nine primers, the primer OPC-18 had
the lowest minimum (0.00) and least average (0.308) genetic similarity values. Hence, it
can be used to distinguish the isolates and fingerprint the B. thuringiensis isolates used in
this study. The isolates were also characterized by PCR-product profiles using cry
specific primers to understand the distribution of cry genes in the isolates. cry\ and cryl
were found in HDl, PI, Dl, PPIO, PPl 1 and PP12; cry% and cry9B were present in HDl,
Dl and PP12; crv9A was found in Bt42, T2 and PP14; cry9C in HDl, PI, Dl, T2 and
PP12; cryW in PP14 and SB. The nem and cyt genes were found in T2 and SI3
respectively. PCR amplification with crylA(c) specific primers revealed the presence of
the gene in HD, PI, Dl, PPIO, PPll and PP12. The isolates PI and Dl had two distinct
plasmid bands of size 18.3kb and 12.8kb and the reference strain HDl had seven distinct
plasmid bands of size 25.33, 17.84, 11.57, 7.08, 4.91, 3.5 and 1.506kb. Plasmid DNA
library of isolate PI was generated in pSK+ vector. Of the 40 clones PCR tested with a
pair of cryl general primers, 26 showed the presence of cryl gene
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