Genetic Engineering of Nicotiana tabacum for Reduced Nornicotine Content

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• 作者：Lily B. Gavilano ; Nicholas P. Coleman ; Leigh-Emma Burnley ; Melissa L. Bowman ; Newton E. Kalengamaliro ; Alec Hayes ; Lowell Bush ; Balazs Siminszky
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2006
• 出版时间：November 29, 2006
• 年：2006
• 卷：54
• 期：24
• 页码：9071 - 9078
• 全文大小：203K
• 年卷期：v.54,no.24(November 29, 2006)
• ISSN：1520-5118

文摘

Nornicotine is an undesirable secondary alkaloid in cultivated tobacco, because it serves as a precursorto 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-demethylaseenzyme during the senescence and curing of tobacco leaves. While the nornicotine content of mostcommercial burley tobacco is low, a process termed "conversion" can bestow considerably increasednornicotine levels in a portion of the plants within the population. Previously, we isolated a nicotineN-demethylase gene, designated CYP82E4, and demonstrated that RNAi-induced silencing ofCYP82E4 and its close homologues is an effective means for suppressing nicotine to nornicotineconversion. In this study, we used real-time polymerase chain reaction to confirm the central role ofCYP82E4 in nicotine N-demethylation by demonstrating that the transcript accumulation of CYP82E4is enhanced as much as 80-fold in converter vs nonconverter tobacco. We also show the design ofan optimized RNAi construct (82E4Ri298) that suppressed nicotine to nornicotine conversion from98% to as low as 0.8% in a strong converter tobacco line, a rate of nornicotine production that isabout 3.6-fold lower than typically detected in commercial varieties. Southern blot analysis showedthat a single copy of the RNAi transgene was as effective in suppressing nornicotine accumulationas multiple copies. Greenhouse-grown transgenic plants transformed with the RNAi construct weremorphologically indistinguishable from the empty vector or wild-type controls. These resultsdemonstrate that the genetic transformation of tobacco with the 82E4Ri298 construct is an effectivestrategy for reducing nornicotine and ultimately NNN levels in tobacco.