Cloning and Transformation of Iranian Firefly Lampyris turkestanicus Luciferase Gene (luc) into Tobacco Plant

“Molecular farming” is the use of plants, and potentially animals, as the means to produce therapeutic and industrial valuable proteins using genetic engineering techniques. Firefly luciferase is one of the most important industrial enzymes which has various applications in biotechnology and molecular biology such as microbial contamination detection by ATP amount, preparation of cancer diagnosis kits, drug screening, biosensors, protein folding and protein-protein interaction examinations, pyrosequencing, etc. luc is also used as an ideal reporter gene in order to optimize gene transformation systems in genetic engineering. In this study, Iranian firefly Lampyris turkestanicus luciferase gene (luc) was cloned and introduced into tobacco plants. The gene was isolated by PCR and appropriate primers, designated according to luc termini, restriction sites and a plant highly expression sequence (Kozak sequence), and then cloned in pCAMBIA1304 vector, under the control of CaMV35S promoter and NOS terminator. The construct was confirmed by several methods: colony PCR, PCR, digestion, sequencing and BLAST analysis. The Agrobacterium–mediated transformation method was used to introduce luc into tobacco genome and putative transgenic plants were selected on the selective medium containing hygromycin and cefotaxim. Finally, transgenic plants were confirmed by PCR analysis on transgenic and control plants’ genomic DNA.