Cloning and Expression Studies of Osmotin-Like Protein Gene from Solanum nigrum in Escherichia coli.

Abstract

Plants face various biotic and abiotic stresses, necessitating the activation of defense mechanisms, including pathogenesis-related (PR) proteins. Osmotin-like proteins (OLPs), belonging to the PR-5 family, play a crucial role in plant defense by enhancing resistance to pathogens and environmental stresses. However, the functional characterization of OLPs remains limited. This study aimed to clone and express the OLP gene from the medicinal plant Solanum nigrum in Escherichia coli to facilitate further functional and structural analyses. The genomic DNA of S. nigrum was isolated from in vitro-cultured plants, and the OLP gene was amplified using primers designed via Primer3 software based on NCBI sequences. Gradient PCR optimization determined the optimal annealing temperature between 58.3 °C and 60 °C. The amplified gene was cloned into the pTZ57R/T vector and transformed into E. coli. Sequencing confirmed a 98% homology with reported OLP sequences. For expression analysis, the gene was subcloned into the expression vector pET15b and transformed into E. coli BL21 (DE3). Induction with 1 mM IPTG at 37 °C for 3 h resulted in the production of a 26 kDa protein, confirmed by SDS-PAGE and Protein Dot Blot analysis using anti-histidine antibodies. The successful cloning and expression of OLP provide a foundation for investigating its role in plant-pathogen interactions and its potential applications in agriculture and medicine. This study contributes to understanding PR proteins and offers insights into their potential for enhancing stress tolerance in crops.