Inducible double-stranded RNA expression activates reversible transcript turnover and stable translational suppression of a target gene in transgenic tobacco.
A binary vector amenable to high-throughput cloning was constructed for ethanol-inducible expression of double-stranded RNA (dsRNA) in plants. Silencing of a transgene encoding beta-glucuronidase (GUS) was then examined at RNA and protein levels in tobacco. Transient gene silencing could be effectively achieved in plants with higher expression levels of alcR (the ethanol sensor) after single application of 1% ethanol (v/v) through root drenching. GUS activities showed more dramatic pattern of loss and recovery in young leaves than in older leaves. Repeated ethanol treatment resulted in extended gene suppression and increased loss of GUS activities. Interestingly, recovery of GUS transcript level is dramatically earlier than that of GUS protein levels as measured by enzyme assays. These observations indicate that dsRNA-mediated gene silencing may occur through more stable translational inhibition in addition to reversible targeted RNA degradation.
Lo
Clive
C
Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
Wang
Nai
N
Lam
Eric
E
D000431:Ethanol / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics*; D020871:RNA Stability / Q000187:drug effects; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics*
D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D000431:Ethanol; D005966:Glucuronidase