Transient RNA silencing of scoulerine 9-O-methyltransferase expression by double stranded RNA in Coptis japonica protoplasts.
RNAi (RNA interference, RNA silencing) is a powerful tool for functional genomics, but the construction of an RNAi vector(s) and the establishment of stable transformants are time-consuming and laborious. Here we report the transient RNAi of endogenous biosynthetic genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica protoplasts. Double stranded (ds) RNA fragments of various lengths prepared from several different positions of the coding sequence of scoulerine 9-O-methyltransferase (SMT) were introduced into C. japonica protoplasts by polyethylene glycol-mediated transformation, and their effects were monitored by reverse transcription-polymerase chain reaction. Substantial silencing of SMT gene expression was obtained by the introduction of these SMT dsRNAs. A significant reduction in SMT protein levels was also observed. The potentials of this transient RNAi system to evaluate the functions of biosynthetic genes in Coptis alkaloid research are discussed.
Dubouzet
Joseph Gogo
JG
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
Morishige
Takashi
T
Fujii
Nanae
N
An
Chung-Il
CI
Fukusaki
Ei-ichiro
E
Ifuku
Kentaro
K
Sato
Fumihiko
F
D001599:Berberine / Q000378:metabolism; D039323:Coptis / Q000201:enzymology / Q000235:genetics*; D017930:Genes, Reporter; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000378:metabolism*
D012330:RNA, Double-Stranded; D001599:Berberine; D008780:Methyltransferases; C093217:S-adenosyl-L-methionine - scoulerine-9-O-methyltransferase