Expression of OmpC gene in Solanum lycopersicum via Agrobacterium-mediated transformation

Abstract

Salmonellosis is a bacterial infection that is caused by Salmonella. Salmonella is a pathogen of animals. This disease spreads to humans mainly from animals i.e. poultry, contaminated food as well as undercooked eggs and meat. Severe Salmonella infections lead to development of colon cancer. Vaccines are used in controlling Salmonella infections. The available vaccines against Salmonella are Ty21a (Vivotif) and Vi CPS. Subunit vaccines are effective against Salmonella infection. Outer membrane protein C (OmpC) is immunogenic protein, found in the outer membrane of Salmonella. OmpC gene is a good candidate for the development of vaccine against Salmonella. Plants are the best source for the development of a cost-effective vaccines where foreign antigens can be expressed. The present research work aimed to optimize an efficient transformation protocol for the stable transformation of Solanum lycopersicum with OmpC gene mediated by Agrobacterium. Sterilization of seeds with 0.1% mercuric chloride followed by their germination on ½ MS media provided good results. Nodal explants were regenerated on full MS media supplemented with 1mg/l BAP and 0.5 mg/l IAA. Different concentrations of antibiotic kanamycin were used for selection of transformed Solanum lycopersicum with optimal concentration being 75 mg/l for nodal explants. Successful transformation with OmpC gene was carried out using above mentioned conditions. 8 minutes infection time and 2 days co-cultivation time provided good results. The transformed explants were grown on selection media supplemented with antibiotic kanamycin. Transformation was confirmed through PCR by using gene specific primers. Transgene expression in tomato plants was analyzed by quantitative real-time PCR (qRT-PCR) in comparison to β-actin gene as control. Protein expression was analyzed through Dot blot analysis. Taken together, the successful expression of OmpC gene in plants may facilitate the development of an edible, cost-effective and subunit vaccine against salmonellosis. Keywords: Solanum lycopersicum, OmpC gene, Salmonellosis, Agrobacterium mediated transformation, qRT-PCR, Dot blot.