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Distribution
vip
Genes, Protein Profiling Determination Entomopathogenic Potential Local Isolates
Bt
16
Table 1 Toxicity of partially purified toxin from different
Bt
isolates for
H. armigera
Sr. No.
Strain
LC
50
(ng/cm
2
) Fiducial limit at 50% LC
90
(ng/cm
2
) Fiducial limit at 90% Slope X
2
1
HD-1
190.42
176.4~222.57
251.98
217.97~466.49
10.53 0.095
2
PDKV-8
161.80
151.06~169.45
194.09
183.03~221.12
16.21 1.604
3
PDKV-21 173.62
163.02~183.18
208.61
194.82~244.91
16.07 0.237
4
NCIM-5110 170.57
157.45~182.86
221.32
200.5~297.72
11.32 0.723
5
NCIM-5132 178.61
166.75~191.97
223.20
203.47~290.86
13.24 0.281
However,
vip3A
(full length) primer showed the
presence of
vip3A
gene in four isolates viz. PDKV-21,
NCIM-5132, NCIM-5130 and HD-1. Percent
abundance data showed 15% abundance of
vip3A
gene
in local
Bt
isolates (Figure 2B). Out of 40 isolates five
isolates viz.
PDKV-3, PDKV-8, NCIM-5110,
NCIM-5132 and HD-1 showed successful amplification
of
vip3Aa1
gene with expected size of 2400 bp.
In total, three types of
vip
genes (
vip1
,
vip2
, and
vip3
)
were identified from the available
Bt
strains with the
primer pairs of
vip1
/
vip2
,
vip3A
partial,
vip3A
full
length, and
vip3Aa1
. Some types of vip gene were not
identified in certain strains.
1.2 SDS PAGE profiling of different
vip
proteins
SDS-PAGE was performed for the total protein
extracted from all 40 supernatants although several
common bands (Figure 3) were detected among some
strains. Overall results clearly demonstrated a
strain-specific pattern of polypeptide secretion in the
culture medium, which reflect final insecticidal
potential of the respective isolate. The PDKV-8 isolate
displayed significant high larvae mortality against
lepidopteran pest (Table 1). PDKV-08 have
particularly relevant band in the position of the
putative VIP3A-like polypeptide (88 kD,) appeared
much more intense than in all others lanes (Figure 3).
Also, strains PDKV-21, NCIM-5132, NCIM-5110 and
HD-1 showed the presence of 88 kD band which
already depicts positive results in molecular screening
for
vip3A
gene specific primer.
1.3 Insect bioassay to study the insecticidal
potential
Four isolates of
Bt
were subjected for insect bioassay
viz.
PDKV-8,
PDKV-21,
NCIM-5110 and
NCIM-5132 with HD-1 as standard reference strain.
Four isolates were selected as a representative samples
as PDKV-08, NCIM-5110 and NCIM-5132 showed
vip3A
and
vip3Aa1
apart from this, PDKV08 also
found to harbour
vip1
/
vip2
. PDKV-21 showed the
presence of very prominent
vip3A
gene (Figure 4).
Figure 3 SDS PAGE electrophoretogram of different
vip
proteins M- unstained protein molecular weight marker
Note: 1: NCIM-2130; 2: NCIM-5110; 3: NCIM-5132; 4: HD-1,
5: PDKV-01; 6: PDKV-21; 7: PDKV-08
Figure 4 Insect bioassay of
vip
toxins against
H. armigera
The molecular analysis and protein electrophoretogram
of
vip
proteins strongly supported the presence of
vip3
proteins in selected isolates to study their insecticidal
activity. Though there was no high magnitude
difference in the LC
50
values. The all test isolates
showed lower LC
50
values than reference strain as
showed in table 1. Insect bioassay is considered as
Bt Research