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Distribution
vip
Genes, Protein Profiling Determination Entomopathogenic Potential Local Isolates
Bt
18
Table 2 The primers for identification of vip genes and finding novel
vip3
-type genes
Target gene
Primer sequence
Amplicon size (bp)
Reference
vip1/vip2
F- 5'-AAATTAGTGATCCGTTACCT TCTT-3'
R- 5'- CAACTTGCTTTTCTTTCCCTTTAT-3'
742
(Shi et al.,
2006)
vip3A
(Partial)
F- 5'-GCACAAGAGCCTTACCAAGT-3'
R- 5'-TTAGCTTAGCCTCATAT-3'
1113
(Bhalla et al.,
2005)
vip3A
(full length)
F- 5'-TAAGAATGCGGCCGCATGAACAAGAATAATACTAA-3'
R- 5'-ACCGCTCGAGTTATCTAATAGAGACATCGT-3'
2370
(Wu et al.,
2004)
vip3Aa1
F- 5'-ATGAACAAGAATAATACTAAATTAAGC -3'
R- 5'-GGTCGACTTACTTAATAGAGACATCG-3'
2400
(Bhalla et al.,
2005)
3.3 Isolation of vegetative insecticidal proteins
According to the method given by Sattar et al. (2008)
vegetative insecticidal proteins were obtained for
insect bioassay. Proteins present in the supernatant
were precipitated with ammonium sulphate
[(NH
4
)
2
SO
4
] (80% saturation) and collected by
centrifugation at 12 000 g for 10 min at 4
℃
. The
pellet was re-suspended in minimum volume of 20
mM of Tris-HCl buffer (pH 7.4) and dialyzed
overnight at 4
℃
against 20 mM Tris HCl (pH 7.4).
The purified proteins were estimated by the Bradford
method (Bradford, 1976) and used for insect toxicity
assay.
3.4 SDS-PAGE profiling of
vip
proteins of
Bt
Vegetative insecticidal proteins were performed by
using 10% Sodium Dodecyl Sulphate Polyacrylamide
Gel Electrophoresis (SDS-PAGE) according to
method given in protocol Sambrook and Russell
(Sambrook and Russell, 2007).
3.5 Insect Bioassay for determining insecticidal
potential of
vip
proteins
Biological activity of v
ip3A
toxins were confirmed by
using standard bioassay procedures. Lepidoptera
bioassays against
H. amigera
neonates were
conducted with diet incorporation method (Yu et al.,
1997).
Bt
subsp.
kurstaki
was used as positive control.
In each experimental set, ten such neonates were
released and observed after each 12 hrs.
4 Conclusions
The study led to the identification of a novel
vip3A
gene, which is known to effective, against
coleopterans. It is necessary to study the effect
against insect pests, clone these genes into a plant
expression vector, transform crop plants and study
their effectiveness against different insect pests.
Authors’ contributions
PS conducted all the research for this paper and prepared the
manuscript. DD participated in bioassays and molecular
characterization. AD coordinated the protein isolation,
purification and profiling experiments. Molecular screening
and characterization was performed in supervision of PJ and
MS. The insect bioassays were carried out under supervision of
NS. MM reviewed the manuscript and coordinated the project.
Acknowledgements
Authors are thankful to Head, Department of Agril Botany and
Officer In-charge, Biotechnology Centre, Dr. Panjabrao
Deshmukh Agricultural University, Akola, for their support.
References
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Bt Research