Cloning of defense response signal proteins genes from Nicotiana sylvestris and construction of RNAi plant expression

In order to analyze the Nicotiana sylvestris N’ gene-mediated tobacco mosaic virus (TMV) resistance signaling pathways and breed plant resistance species to TMV, eight genes including PAD4, EDS1, Rar1, Sgt1, NPR1, Jar6, COI1, CTR1 were selected as candidate genes. Total RNA of N.sylvestris was extracted as templates and eight genes fragments were cloned by RT-PCR. After digesting with appropriate enzyme, ligase and transformation, RNAi vectors of the eight genes were successfully constructed. The RNAi vectors can be used for genetic transformation experiment after their structure were confirmed by the same enzyme digest. These vectors laid foundation for analyzing the N’ gene-mediated TMV resistance signaling pathways.