Abstract:

RPP13 is a typical R gene that can induce plant immune response by recognizing of the effectors.The transcriptome sequencing analysis of five potato cultivars infected with Potato spindle tuber viroid (PSTVd)showed that RPP13 genes were up-regulated in the five potato cultivars infected with PSTVd.In order to study whether this gene was related to PSTVd resistance in potato,DNAMAN 8.0 software was used to perform sequence alignment analysis on RPP13 upregulated gene sequences obtained by transcriptome sequencing.The common conserved region was selected as the silencing object,and a stem-loop structure inverted repeat with the conserved region as the arm and the intron from somatic embryogenesis receptor-like kinase 1 (SERK1)(Accession number EF175216)as the loop was inserted in plant expression vector pROKⅡ to construct RNAi vector named pROKⅡ-RPP13.The pROKⅡ-RPP13 vector was transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method,and the potato cultivar Minfenghong was transformed by Agrobacterium tumefaciens mediated genetic transformation method. Healthy potato leaves of 28 days of seedling age were used as materials. After 2 days of pre-culture, 10 minutes of infection, 2 days of co-culture under dark conditions, roots and buds were induced by the resistance screening medium. The regenerated plants were screened by PCR, and three positive transgenic plants were obtained.Using efla gene of potato as the reference gene,Real-time RT-PCR was used to detect the expression of RPP13 gene in potato transformed plants.The results showed that the expression level of RPP13 gene in the three transgenic plants obtained was significantly different from that in the control without transgenic.In the obtained RNAi transgenic potato plants,the expression level of RPP13 gene was decreased by 35%—60%.

Key words: Potato, Genetic transformation, RNAi, RPP13 gene, Fluorescence quantitative PCR