Construction Of RNA Interference Vector Of Sweet Potato Chlorotic Stunt Virus And Genetic Transformation Of Tobacco

Sweet potato chlorotic stunt virus(SPCSV)is one of the most important viruses infecting sweet potato. SPCSV can co-infect sweet potato with other viruses and cause synergistic effect. RNA interference has become an effective method to develop transgenic antiviral crops. Besides, a novel transgenic antiviral strategy based on ami RNA( artificial micro RNA) has emerged in recent years. This technique is characterized as high stability, effectiveness and safety. A ds RNA-specific endonucleases belonging to RNaseⅢ(RNase3)is encoded by SPCSV and speculated as a gene silencing suppressor of SPCSV. Therefore, this study is trying to explore the biological function of RNase3 and obtain transgenic plants of tobacco with virus resistance. The main results were summarized as follows:(1)The complete sequence of RNase3 gene was chosen to construct plant expression vector and RNA interference vector. The gene was cloned reversely and repetitively on both sides of the intron of p Blue Ni RIntron. The fragment of RNase3-Intron-RNase3 was then inserted into p ROK219 between 35 S promoter and NOS terminator. Finally the fragment of 35S-RNase3-Intron-RNase3-NOS was cloned into p Bin19,to construct RNA interference vector p Bin19 Ri.(2)Similarly,RNase3 gene was inserted into p ROK219 between 35 S promoter and NOS terminator, and then cloned into p Bin19 to construct plant expression vector p Bin19 R3. These two vectors were further confirmed by PCR analysis and enzymatic digestion.(3)RNase3-specific ami RNA was designed by WMD3 and selected using ami RNA design principle. The precursor of the Arabidopsis mi R159a(pre-mi R159a) was modified as pre-ami R159a-R3 which can produce ami RNA targeting RNase3. It was artificially synthesized and cloned into PBI121,an ami RNA interference vector PBI121 ami R was successfully constructed.(4)All the vectors were seperately transformed into Agrobacterium tumefaciens strainEHA105 by freeze-thaw method. The vectors were transformed into tobacco variety K326 by Agrobacturium-mediated leaf disk transformation method. As the result, 14 regenerated plants were obtained for p Bin19 Ri,14 regenerated plants were obtained for p Bin19 R3,and 18 regenerated plants were obtained for PBI121 ami R.