title: Genetic engineering of Nicotiana tabacum for reduced nornicotine content.
abstract: Nornicotine is an undesirable secondary alkaloid in cultivated tobacco, because it serves as a precursor to N'-nitrosonornicotine (NNN), a tobacco-specific nitrosamine with suspected carcinogenic properties. Nornicotine is produced through the oxidative N-demethylation of nicotine by a nicotine N-demethylase enzyme during the senescence and curing of tobacco leaves. While the nornicotine content of most commercial burley tobacco is low, a process termed "conversion" can bestow considerably increased nornicotine levels in a portion of the plants within the population. Previously, we isolated a nicotine N-demethylase gene, designated CYP82E4, and demonstrated that RNAi-induced silencing of CYP82E4 and its close homologues is an effective means for suppressing nicotine to nornicotine conversion. In this study, we used real-time polymerase chain reaction to confirm the central role of CYP82E4 in nicotine N-demethylation by demonstrating that the transcript accumulation of CYP82E4 is enhanced as much as 80-fold in converter vs nonconverter tobacco. We also show the design of an optimized RNAi construct (82E4Ri298) that suppressed nicotine to nornicotine conversion from 98% to as low as 0.8% in a strong converter tobacco line, a rate of nornicotine production that is about 3.6-fold lower than typically detected in commercial varieties. Southern blot analysis showed that a single copy of the RNAi transgene was as effective in suppressing nornicotine accumulation as multiple copies. Greenhouse-grown transgenic plants transformed with the RNAi construct were morphologically indistinguishable from the empty vector or wild-type controls. These results demonstrate that the genetic transformation of tobacco with the 82E4Ri298 construct is an effective strategy for reducing nornicotine and ultimately NNN levels in tobacco.
  
  
  BACKGROUND
  Alkaloid; cytochrome P450; gene silencing; nicotine N-demethylase; N'-nitrosonornicotine; plant genetic engineering; metabolic engineering; Nicotiana tabacum L.; real-time PCR; RNA interference; tobacco-specific nitrosamines.
authors.affiliation: Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546, USA.
authors.forename: Lily B
authors.initials: LB
authors.lastname: Gavilano
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Coleman
authors.forename: Leigh-Emma
authors.initials: LE
authors.lastname: Burnley
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Bowman
authors.forename: Newton E
authors.initials: NE
authors.lastname: Kalengamaliro
authors.forename: Alec
authors.initials: A
authors.lastname: Hayes
authors.forename: Lowell
authors.initials: L
authors.lastname: Bush
authors.forename: Balazs
authors.initials: B
authors.lastname: Siminszky
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D017931:DNA Primers; D006801:Humans; D009538:Nicotine / Q000031:analogs & derivatives* / Q000737:chemistry; D018515:Plant Leaves; D030821:Plants, Genetically Modified; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics*
chemical_list: D017931:DNA Primers; D009538:Nicotine; C005311:nornicotine; D003577:Cytochrome P-450 Enzyme System