Abstract: In order to obtain potato plants which could be resistant to multiple viruses (viroid) simultaneously, P25, HC-Pro, and Virp1 gene targeting PVX, PVY, and PSTVd proteins were cloned, respectively. Three types of amiRNAs targeting sequences encoding the silencing suppressor P25, HC-Pro and Virp1 were designed by using Arabidopsis thaliana miR159a as backbone. These three amiRNA sequences were connected by Overlapping PCR. The synthetic of P25, HC-Pro and Virp1 gene was inserted into the expression vector pCAMBIA1300-221 to form p1300-221-preamiR-P25-HCPro-Virp1, and the vector was verified by PCR and restriction enzyme digestion. Pre-amiR-P25-HCPro-Virp1 was transformed into minituber of potato cultivar Favorita by Agrobacterium tumefaciens infection. 15 transformed plants were obtained through regenerating, pressure screening and differentiation.PCR results showed that 10 of them were detected the same size fragment (729 bp) as the target gene. Further qRT-PCR testing confirmed that amiR-P25-HCPro-Virp1 gene was expressed in 10 transformed plants, and the relative expression was between 7.68-21.37. Inoculated the T₀ transgenic plants with the mixture virus of PVX, PVY and PSTVd by friction into the leaves of the plants at the 6-8 leaf stage. The plant growth was observed after 20 days and the results showed that the control plants were severely susceptible, with symptoms such as short plants and mottled leaves, while the transformed plants did not show infection symptoms, and growth normally. There was no virus detected by RT-PCR either. This indicated that the transfected amiR-P25-HCPro-Virp could be stably expressed in the transformed plants, and the transformed plants were resistant to PVX, PVY and PVSTd viruses (viroid). Transformed plants resistant to PVX, PVY and PVSTd were obtained, and the resistance was significant, which provided new genetic resources for potato virus-resistant breeding.

Key words: Potato, amiRNA, PVX, PVY, PSTVd

CLC Number: 

• S532