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Bt Research
17
very important and confirmation studies with respect
to the insecticidal protein.
2 Discussions
The present investigation was carried out to
characterize
Bt
isolates obtained from native
ecological niche for the presence of different
vip
genes
and also to explore the possibility of getting any new
vip
gene (s). The recent insect resistance scenario
prompted us to attempt this study. It is first attempt to
study the distribution of different
vip
genes in local
Bt
isolates from Vidarbha region of Maharashtra (India).
It has been reported that
vip
proteins account for about
15% of the
Bt
strains (30). With the three pairs of
primers, we found that the average rates of
vip3A
(12.5%) was the most and the average rates of
vip1
/
vip2
(10%) were similar with the results reported
by Herna´ndez-Rodrı´guez et al. (2009). Whereas in
present finding, abundance of
vip
like genes are not
more than 12.5%. Other researchers have reported
such lower frequencies of
vip
genes: 23.2% (Rice,
1999), 2% (Selvapandiyan et al., 2001) and 33.3%
(Bhalla et al., 2005). However, Loguericio et al.
(Loguercio et al., 2002) and Fang et al. (Fang et al.,
2007) reported the presence of
vip
like genes in all the
isolates, Beard et al. (Beard et al., 2008) also showed
the similar trends. Yu et al. (2010), found 67.4%
abundance of
vip3
type of genes in from Sichuan
basin in China. Similarly, Liu et al. (2008), found
63.03%. High abundance of
vip
genes in these reports
are contradictory to our findings. This might be due to
difference in habitat, geographical location and other
environmental conditions.
The main purpose of our research was finding
insecticidal proteins for Lepidoptera. Therefore, this
study concentrated on finding novel
vip3
-type genes.
We have found five novel
vip
genes, which could
enrich the available categories of insecticidal genes.
However, our isolates had very few novel vip genes.
The reason why there were only five novel
vip3
genes
in screened
Bt
strains might be that the
vip
genes
(three groups of
vip
genes have been researched) were
more conserved than
cry
genes (67 groups of
cry
genes have been researched), and the referenced
method may not be the best way to probe for
vip
genes
(Fang et al., 2007). Therefore, we should find better
ways to identify novel genes.
To find toxicity of
vip3
for
H. armigera
, the four
novel vip proteins of the screened
Bt
strains were
assayed with
H. armigera
. The partially purified
proteins of
Bt
were bioassayed against
H. armigera
with positive and negative controls. The bioassay
results indicated that the 4 screened
Bt
samples really
include novel
vip3
genes. The presence of different
insecticidal genes detected, may account for variation
in the toxicity of these isolates. According to
Monnerat et al. (Monnerat et al., 2007) the greater
potency of these isolates than
Bt-kurastaki
HD 1
against the target insects may be due to (i) variation in
the number of genes harbour by the isolates, (ii)
higher level of accumulation of toxins, (iii) the
presence of other Bt toxin genes that are not detected
by the primers used in the screening (iv) combination
of all these factors.
3 Materials and Methods
3.1 Local
Bt
strains used in present investigation
40 local
Bt
strains were subjected for molecular
screening. Out of these, 28 isolates (PDKV-01 to 28)
were isolated by Kedar, S.B. 2011 from the campus of
Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola
and Nagpur campus of Dr. PDKV. Reference strain
HD1 was kindly supplied by Dr. Donald Dean
(
Bacillus
Genetic Stock Centre, Columbus, Ohio).
Similarly eleven different subspecies of Bt were
obtained from NCIM (National Center for Industrially
useful Microbes (NCIM)), NCL, Pune.
3.2 Total genomic DNA isolation and PCR
screening of
Bt
isolates
Genomic DNA was isolated from the
Bt
isolates as per
the method given by Kate Wilson (Wilson, 1997). All
PCR amplifications were performed by using the
gradient thermal cycler (EP gradient, Eppendorf).
Isolates were tested for the presence of
vip
genes with
primers indicated in Table 2.
After the positive screening of
Bt
isolates for the
presence of
vip
genes attempts are made to harbour
the full length
vip
genes. Three primers are designed
on the basis of full length
vip
gene sequences to
amplify the coding DNA sequences (cds) of
vip
genes.
PCR results were analysed by using 1.2% agarose gel.
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