RFLP analysis of cry1 and cry2 genes of Bacillus thuringiensis isolates from different regions of India

Abstract

The PCR-RFLP method has been useful for detection of
known genes and identification of novel genes. In the
present study, degenerate primers were designed from
five groups of cry1 genes for PCR-RFLP analysis. Bacillus
thuringiensis (Bt) isolates from different regions were
evaluated for PCR amplification of various cry1 genes
using newly designed primers and cry2 genes using
reported primers. PCR analysis showed an abundance of
cry1A genes and especially cry1Ac genes in isolates from
all regions. RFLP analysis revealed the presence of multiple
cry1A genes in isolates from central and southern regions.
Unique digestion patterns of cry1A genes were observed in
isolates from each region. Few of the isolates represented a
digestion pattern of cry1A genes that did match to any of
the known cry1A genes. RFLP analysis suggested an
abundance of cry2Ab along with a novel cry2 gene in Bt
isolates from different regions of India. Sequence analysis
of the novel cry2 gene revealed 95% sequence identity to
cry2Ab and cry2Ah genes. Phylogenetic analysis revealed
that the novel cry2 gene could have diverged earlier than
the other cry2 genes. Our results encourage finding of
more diverse cry2 genes in Bt isolates. Rarefaction analysis
was used to compare cry1A gene diversity in isolates from
different soil types. It showed a higher degree of cry1A
gene diversity in isolates from central region. In the
present study, we propose the use of novel degenerate
primers for cry1 genes and the PCR-RFLP method using
a single enzyme to distinguish multiple cry1A and cry2
genes as well as identify novel genes.