Elucidation of Plant RNAi mechanism and its application to gene regulation.

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1.Depevolpment of novel trangent RNAi system by direct introduction of dsRNA into plant protoplast.An practical trangent RNAi assay system was developed. Synthetic dsRNA that were corresponding to the target gene sequences were introduced into Arabidopsis protoplast cells by PEG associated lipofection. The target gene expression in the cells to which dsRNA was introduced was effectively repressed. To verify the effectiveness of the system, several endogeneous genes of Arabidopsis, such as PAT1, the gene of phosphoribosylanthranilat transferase, sdh2-J and sdh2-1, the gene of succinate dehydrogeneas were targeted. The system was proved to be effective for the selective gene silencing using 3'UTR as RNAi target sequences. In addition, the system was applied to seeking the gene for berberin biosynthesis in Coptis japonica by observing the loss of function by the introduction of the dsRNA corresponding to the candidate genes.2.An application of RNAi for plant metabolic engineering.RNAi was applied to modulation of the metabolic gene concerning biosynthesis of petal color pigment in plant. The gene of chalcone synthase, the key enzyme of anthocyanin biosynthesis, in trenia (Torenia hybrida) was targeted. The inner part of CDS and the 3'UTR of torenia CHS gene were used for RNAi sequences. Both transformant were fully elucidated for gene expression, petal color phenotype, anthocyanin production. The gene specific repression by the system using 3'UTR was verified.