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Screening of vip1/vip2 binary toxin gene and its isolation and cloning from local Bacillus thuringiensis isolates

Prashant R. Shingote, Mangesh P. Moharil^*, Dipti R. Dhumale, Pravin V. Jadhav, Niraj S. Satpute, Mahendra S. Dudhare

 
ABSTRACT:     Efforts were carried out to isolate vegetative insecticidal protein genes from local isolates of Bacillus thuringiensis. The study focused on vip1/vip2 binary toxin, considering its insecticidal potential against coleopteran and hemipteran pests. Thirty nine B. thuringiensis local strains and one standard reference strain (HD 1) were screened for the presence of vip1/vip2 gene by using a PCR approach. Among 39 isolates only four isolates (PDKV-08, PDKV-27, PDKV-28 and NCIM-5112) showed the presence of the desired gene. SDS-PAGE screened profiling of the isolates showed the presence of 95 kDa and 50 kDa protein which confirmed our PCR study. For further characterization, the vip1/vip2 gene was cloned from the PDKV-08 isolate by using the pJET1 cloning vector. Sequence homologous analysis confirms the presence of the vip1/vip2 gene. A further BLAST analysis also revealed that the isolated vip1/vip2 gene is highly conserved and showed a maximum of 88% sequence homology with existing vip1/vip2 genes. Insect toxicological potential was also elucidated by performing bioassays of PDKV-08 supernatant proteins against the coleopteran store grain pest, Sitophilus zeamais. The results from a bioassay revealed 60% mortality.

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* Corresponding author, E-mail: mpmoharil@gmail.com

Received 14 Sep 2012, Accepted 27 Aug 2013