The Establishment And Optimization Of Nicotiana Sanderae VIGS System

Nicotiana sanderae, belonged to Nicotiana, Solanaceae, is the hybrid strain of Nicotiana forgetiana with red and non-fragrant flowers and Nicotiana alata with white and fragrant flowers. It is quite suitable for study the function of flower color and flavor genes, owing to its beautiful color and flavor. In order to build up a technology platform for large-scale verificating the function of flower color and flavor genes in N. sanderae, we used the recombinant viral vector Tobacco Rattle Virus (TRV), and took Phytoene desaturase gene and chalcone synthase gene as reporter genes, initially built and optimized the VIGS system of N. sanderae. The results were as follow:1、In the experiment of silencing of the reporter gene PDS, we used root to infect N. sanderae’s0.5’～1cm young roots were infected with root absorption method. The survival rate of the seeds collected from the infected plants was below30%after dibble seeding, and, during the whole life cycle of the survivors, the expected phenomenon of leave photobleaching had never appeared, which suggested that root absorption method was unsuitable for induction of gene silencing in N. sanderae. However, when the N. sanderae grew up to five or six leaf stage, the reverse side of inner wheel leafs were infected by the method of dorsal injection (syringe without needle). In this process,200～400μl OD600=1.1agrobacterium were injected into leaves, and injected plants were cultured in light incubator, the condition was set to16h25℃and8h18℃under full darkness for one day, then set to16h25℃under light and8h18℃under darkness for routine maintenance.25days later, in the treatment group,80%of plants exhibited the expected leaf photobleaching.2、"siRNA Scan "was used to screen the CHS gene fragment that could effectively produce21nt siRNA. Then ligation-independent cloning method was used to construct recombinant viral vector pTRV2-CHS, the positive rate reached100%.3、In the experiment of silencing the report gene CHS, VIGS was used to do the leaf infection.30or45days after infection, when the autumn came, and the temperature had never exceeded28℃, the plants were moved from light incubator to greenhouse in which3hours’lighting was added after sunset. After conventional cultivation,60%of the plants exhibited the expected phenomenon that corolla became partly white. RT-PCR test showed that the expression of CHS gene was really down-regulated. However, the treatment groups through apical meristem infection didn’t exhibited the expected phenotype.