Cloning and Transformation of Bacteriophage T7 RNA Polymerase in Tobacco

Molecular farming refers to the production of pharmaceutical proteins and industrial enzymes in plants by genetic engineering. T7RNA Polymerase is one of the most important enzymes which is used in various fields of biotechnology and molecular biology to produce RNA and study the structure and function of RNA in vitro. The in vitro synthesized RNA is employed in hybridization, microarray, anti-sense RNA and RNAi experiments. This enzyme has also applications in expression systems depending on the T7 promoter and terminator. T7RNA polymerase is a bacteriophage enzyme which acts mainly in the cytoplasm. This study aimed to clone and transform eukaryotic cells with the T7RNA Polymerase gene for production of T7 polymerase in tobacco plants. Specific primers with proper restriction enzyme sites were designed corresponding to the ends of the T7RNA polymerase gene and the gene was isolated and cloned into the plant expression vector, pCAMBIA1304. Successful of gene insertion was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The recombinant construct was transformed into Agrobacterium tumefaciens LBA4404 strain for tobacco transformation. The transgenic tobacco plants were regenerated on selective media containing hygromycin and cefotaxime antibiotics. The presence and expression of the T7RNA polymerase gene in the transgenic plants was confirmed by PCR and RT-PCR techniques.