Abstract
A high through put transient RNAi assay system would be useful for plant metabolic profiling. We developed a practical system using protoplast derived from Arabidopsis thaliana leaf and cell culture T87. A dsRNA including a partial sequence of a target gene was prepared by in vitro transcription system. Introduction of dsRNAs by transfection method with PEG and an expression level of the target gene was evaluated by real time RT-PCR. The two endogenous homolog genes sdh2-1 and sdh2-2 were chosen as targets. Their CDS are highly homologous but their 3'UTR aren't. In case of CDS targeting, both genes were down regulated. However, the 3'UTR targeting led a specific suppression of the corresponding gene. Transformation efficiency was up to 80% and the duration was around five days. A metabolic profiling of Coptis japonica tissue culture cell is being under study.
