Elucidation of the functions of genes central to diterpene metabolism in tobacco trichomes using posttranscriptional gene silencing.
The functions of two key, trichome-expressed genes were assessed using different posttranscriptional gene silencing strategies (PTGS). Efficient RNA interference (RNAi) revealed the function of a cembratriene-ol (CBT-ol) cyclase gene responsible for conversion of geranylgeranyl pyrophosphate to CBT-ols, and verified the function of a P450 gene responsible for conversion of CBT-ols to CBT-diols. CBT-diols are abundant diterpenes that comprise about 60% and 10% of trichome exudate weight and leaf dry weight, respectively, in Nicotiana tabacum, T.I. 1068. The relative efficiencies and levels of suppression using antisense (AS), sense co-suppression (S), and RNAi were compared for these two genes. With a partial cDNA of the P450 gene, the suppression efficiencies (percent of primary transformants with high CBT-ols/CBT-diols) were low, 3.3% for AS and 0% for S plants. In contrast, using RNAi with a partial gene sequence, a knockdown efficiency of about 45% was achieved. For the CBT-ol cyclase gene, no suppression was observed using partial cDNAs in AS or S orientations, while RNAi with a partial gene sequence yielded an efficiency of about 64%. The efficiencies of gene silencing using full-length coding regions of both genes in AS and S orientations were </=20%. Our results identify the function of a CBT-ol cyclase gene and demonstrate the efficacy and superiority of RNAi for assessing the functions of two trichome-specific genes that encode enzymes having widely different functions.
Wang
Erming
E
Plant Physiology/Biochemistry/Molecular Biology Program, Agronomy Department, 200L THRI, University of Kentucky, Lexington, KY40546-0236, USA.
Wagner
George J
GJ
D022081:Cell Surface Extensions / Q000235:genetics* / Q000378:metabolism; D002855:Chromatography, Thin Layer; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004224:Diterpenes / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D004224:Diterpenes; D016376:Oligonucleotides, Antisense; D010940:Plant Proteins; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System