Liedschulte Verena, Schwaar Joanne Deborah, Laparra Hélène, Vuarnoz Aline, Philippon Bérangère, Bakaher Nicolas, Sierro Nicolas, Bovet Lucien, Lang Gerhard, Goepfert Simon
PMI R&D, Philip Morris Products SA, (part of Philip Morris International Group of Companies), Neuchâtel, Switzerland.
Phytochemistry. 2016 Nov;131:9-16. doi: 10.1016/j.phytochem.2016.08.004. Epub 2016 Sep 3.
In the tobacco plant, nicotine N-demethylase enzymes (NND) belonging to the cytochrome P450 family catalyse the conversion of nicotine to nornicotine, the precursor of the carcinogenic tobacco-specific N-nitrosamine, N-nitrosonornicotine. To date three demethylase genes, namely CYP82E4, CYP82E5 and CYP82E10, have been shown to be involved in this process, while the related CYP82E2 and CYP82E3 genes are not functional. We have identified a further gene named CYP82E21 encoding a putative nicotine N-demethylase closely related to the CYP82E genes. The CYP82E21 gene was found in all Nicotiana tabacum cultivars analysed and originates from the tobacco ancestor Nicotiana tomentosiformis. We show that, in contrast to all other previously characterized NND genes, CYP82E21 is not expressed in green or senescent leaves, but in flowers, more specifically in ovaries. The nicotine N-demethylase activity of CYP82E21 was confirmed by ectopic expression of the coding sequence in a tobacco line lacking functional CYP82E4, CYP82E5 and CYP82E10 genes, resulting in an eightfold increase of nicotine demethylation compared to the control plants. Furthermore, nornicotine formation can be reduced in ovaries by introducing a CYP82E21-specific RNAi construct. Together, our results demonstrate that the CYP82E21 gene encodes a functional ovary-specific nicotine N-demethylase.
在烟草植株中，属于细胞色素P450家族的尼古丁N-脱甲基酶（NND）催化尼古丁转化为去甲烟碱，而去甲烟碱是致癌性烟草特有N-亚硝胺N-亚硝基去甲烟碱的前体。迄今为止，已证明有三个脱甲基酶基因，即CYP82E4、CYP82E5和CYP82E10参与此过程，而相关的CYP82E2和CYP82E3基因无功能。我们鉴定出了另一个名为CYP82E21的基因，它编码一种与CYP82E基因密切相关的假定尼古丁N-脱甲基酶。在所有分析的烟草栽培品种中均发现了CYP82E21基因，其起源于烟草祖先绒毛状烟草。我们发现，与所有其他先前表征的NND基因不同，CYP82E21不在绿色或衰老叶片中表达，而是在花中表达，更具体地说是在子房。通过在缺乏功能性CYP82E4、CYP82E5和CYP82E10基因的烟草品系中异位表达编码序列，证实了CYP82E21的尼古丁N-脱甲基酶活性，与对照植株相比，尼古丁脱甲基作用增加了八倍。此外，通过引入CYP82E21特异性RNAi构建体，可以减少子房中去甲烟碱的形成。总之，我们的结果表明，CYP82E21基因编码一种功能性子房特异性尼古丁N-脱甲基酶。
