Identification of regulatory protein genes involved in alkaloid biosynthesis using a transient RNAi system.

RNAi (RNA interference, RNA silencing) is a powerful tool in functional genomics. We report here the use of transient RNAi to isolate regulatory factor genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica protoplasts. Double-stranded (ds) RNAs prepared against candidate regulatory factors, which were predicted from an EST library, were introduced into C. japonica protoplasts by polyethylene glycol (PEG)-mediated transformation, and their effects were monitored by real-time reverse transcription (RT)-polymerase chain reaction (PCR). The potential of this transient RNAi system to characterize the functions of regulatory factor genes in alkaloid research is discussed.