Cloning and expression studies of osmotin like protein gene from Solanum nigrum in Escherichia coli

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Abstract
Plants usually face different types of stresses both biotic and abiotic. To combat against these stresses, they have defensive proteins. That only induce when there is a need of them. Plant pathogenesis related proteins (PR) is a group of proteins that help plants to fight against the stresses. Osmotin like protein is one of them and belongs to superfamily PR 5. In this study, OLP gene was isolated from the DNA of a medicinal plant Solanum nigrum. The plant was cultured in tissue culture laboratory under specific conditions. Plant genomic DNA was isolated by following a modified protocol. To isolate the gene, primers set was designed by Primer3 software with retrieved gene sequence from NCBI data base. The OLP gene was amplified by gradient PCR at specific set conditions. The annealing temperature range was set at 50°C-60°C. The primers to be optimized showed the optimum annealing temperature at 58.3°C to 60°C. The gel eluted amplified PCR product was cloned in cloning vector pTZ57R/T by using a cloning kit. The transformed plasmid DNA was sequenced to confirm the insertion of gene. The homology of sequenced gene was 98% with the reported sequences. To study the expression of the gene, the OLP gene was cloned in an expression vector pET15b. The construct was transformed into BL21 DE3 (E. coli strain). The expression of protein was analyzed through12% SDS- PAGE after inducing the cells for 3 h at 37°C with 1mM final concentration of IPTG as inducer. The clear difference was observed between induced and un-induced cells through protein profile. The induced OLP was at the right size of 26 kDa. Protein inclusions were made and protein blotting was done by Protein Dot Blot method. By using anti histidine antibodies and color reaction, the clear result of induced osmotin like protein was observed.

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