RNAi suppression of the morphine biosynthetic gene salAT and evidence of association of pathway enzymes.
Papaver somniferum L. was transformed with an RNAi construct designed to reduce transcript levels of the gene encoding the morphine biosynthetic enzyme, salutaridinol 7-O-acetyltransferase (SalAT). RNA interference of salAT led to accumulation of the intermediate compounds, salutaridine and salutaridinol, in a ratio ranging from 2:1 to 56:1. Along the morphine biosynthetic pathway, salutaridine is stereospecifically reduced by salutaridine reductase (SalR) to salutaridinol, which is subsequently acetylated by SalAT. SalAT transcript was shown by quantitative PCR to be diminished, while salR transcript levels remained unaffected. Yeast two-hybrid and co-immunoprecipitation analyses indicated an interaction between SalR and SalAT, which suggested the occurrence of an enzyme complex and provided an explanation for the unexpected accumulation of salutaridine. Decreased concentrations of thebaine and codeine in latex were also observed, while the morphine levels remained constant compared to concentrations found in untransformed control plants.
Kempe
Katja
K
Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA.
Higashi
Yasuhiro
Y
Frick
Susanne
S
Sabarna
Khaled
K
Kutchan
Toni M
TM
D000123:Acetyltransferases / Q000235:genetics*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D009020:Morphine / Q000096:biosynthesis*; D010207:Papaver / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D021241:Spectrometry, Mass, Electrospray Ionization; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D009020:Morphine; D000123:Acetyltransferases