Construction of transformation vector carrying enviromental frendly selection marker and virus resistant gene
Keywords:Nicotiana tobacum, RNA interference (RNAi), manA gene, positive selection, tobacco mosaic virus.
The design of a plant transformation vector carrying environmental friendly selection marker gene for production of bio-safety transgenic plants will promote the commercialization of transgenic products. In this study, a binary transformation vector pK7M/TMV-CPi containing TMV-CPi RNAi construct and manA gene was constructed. TMV-CPi RNAi construct is a inverted repeat transgene cassette, containing partial coat protein gene sequence of tobacco mosaic virus (TMV), that can silence the CP gene of TMV in the TMV resistant transgenic plants. ManA gene encoding phosphomannose isomerase (PMI) enzyme is a positive selectable marker which allows using mannose for selection. With this vector, the TMV-CPi RNAi construct and manA gene have been transformed into two Nicotiana tabacum cv. K326 and C9-1 via Agrobacterium tumefaciens. There were 100 T0 transgenic tobacco lines (64 of K326 and 34 of C9-1 transgenic tobacco lines) which growing well on mannose selective medium were analyzed by artificial inoculation with TMV and PCR. The result showed that 64.3% (18 of 28) and 58.3 % (7 of 12) of K326 and C9-1 transgenic tobacco lines, respectively, were completely resistant to TMV and positive PCR with transgene.