Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara, 630-0192 Japan.
Plant Cell Physiol. 2012 Dec;53(12):2038-46. doi: 10.1093/pcp/pcs139. Epub 2012 Oct 3.
Nornicotine is formed from nicotine by nicotine N-demethylase, a CYP82E family monooxygenase, and accumulates to high levels in some tobacco (Nicotiana tabacum) cultivars and many wild Nicotiana species. Nicotiana langsdorffii does not form nornicotine, whereas the closely related species N. alata accumulates this alkaloid abundantly. We show here that the two nicotine N-demethylase genes in N. langsdorffii have been inactivated by different molecular mechanisms. We identified four N. alata CYP82E genes that encode functional nicotine N-demethylases. In N. langsdorffii, however, one CYP82E gene encoding a functional enzyme was not expressed at all, whereas the other was weakly expressed but contained a one-nucleotide deletion in the first exon, yielding a truncated protein. Expression analysis of interspecific F(1) hybrids between N. alata and N. langsdorffii indicated that cis-acting polymorphisms abolish expression of the otherwise functional CYP82E gene in N. langsdorffii. Segregation analysis of tobacco alkaloids and individual CYP82E alleles in F(2) progeny revealed that duplicated CYP82E genes in both species are genetically linked, and provide genetic evidence that CYP82E genes are solely responsible for nornicotine formation in these wild Nicotiana species.
假木贼碱是由尼古丁 N-去甲基酶（一种 CYP82E 家族单加氧酶）从尼古丁形成的，并在一些烟草（Nicotiana tabacum）品种和许多野生烟草物种中积累到高水平。诺氏烟草（Nicotiana langsdorffii）不会形成假木贼碱，而亲缘关系密切的物种 N. alata 则大量积累这种生物碱。我们在这里表明，诺氏烟草中的两个尼古丁 N-去甲基酶基因已被不同的分子机制失活。我们鉴定了 N. alata 中的四个 CYP82E 基因，这些基因编码具有功能的尼古丁 N-去甲基酶。然而，在 N. langsdorffii 中，一个编码功能酶的 CYP82E 基因根本没有表达，而另一个基因则表达较弱，但在第一个外显子中含有一个单核苷酸缺失，导致截短的蛋白质。N. alata 和 N. langsdorffii 种间 F(1)杂种的表达分析表明，顺式作用多态性使 N. langsdorffii 中原本有功能的 CYP82E 基因失活。F(2)后代烟草生物碱和单个 CYP82E 等位基因的分离分析表明，两个物种中的重复 CYP82E 基因在遗传上是连锁的，并提供了遗传证据表明 CYP82E 基因是这些野生烟草物种中假木贼碱形成的唯一原因。
