Construction and Genetic Transformation of Potato Virus and Viroid Multi-resistant amiRNA Binary Expression Vectors

Potato viruses and viroid disease had seriously affected the production of potato in China. In order to cultivate virus and viroids antiviral new materials of potato. In this study, we utilized the strategy of artificial
miRNA (amiRNA) with the silencing suppressor P25 protein and HC-pro protein which encoded by potato viral pathogens potato virus X (PVX) and potato virus Y (PVY) as the target, and we utilized 5'RACE technique to forecast the potential shear hot spots of P25 gene and HC-Pro gene. Then we used overlapping PCR technology, and also using Arabidopsis thaliana miR159a precursor as skeleton to design specific primers, so we could construct amiRNA expression vector which was respectively targeting P25 and HC-Pro genes. We constructed the amiRNA expression vector targeting the endogenous gene Virp1 of potato at the same time. And we could obtain amiRNAs three by seriesing three amiRNA expression vector which were construction completed. And then we transferred into the Agrobacterium strain EHA105 respectively, we used Agrobacterium mediated transformation of potato varieties Kexin13. Detected by PCR, the results showed that the target gene was successfully transferred into potato.