烟草重要基因篇：3.烟草烟碱合成代谢相关基因

张洪博

（西南大学农学与生物科技学院，重庆 400715）

烟碱（nicotine），即尼古丁，是烟草生物碱（包括烟碱、降烟碱、新烟草碱和假木贼碱等）的一种，约占烟草生物碱总含量的90%～95%[1]。烟叶烟碱含量占叶片干重的0.6%～3.0%，是烟草和卷烟质量的一项重要指标。对烟碱代谢的分子遗传学研究可以揭示烟碱代谢累积的分子机制，并为烟碱含量及烟碱成分调节相关的育种工作提供理论基础。近年来，有关烟碱合成、转运及转化的一些重要基因已被陆续克隆，对烟碱合成代谢机理研究和烟草遗传育种工作产生了重要推动作用。

1 烟碱合成相关基因

烟碱分子由一个吡咯烷环和一个吡啶环构成，在烟草根部合成，通过木质部向地上部运输，在烟草植株的叶片中含量最高，茎部含量最低[2-3]。

烟碱吡咯烷环由氮代谢中形成的腐胺合成。腐胺可通过精氨酸脱羧酶（ADC，arginine decarboxylase）催化精氨酸脱羧形成，或由鸟氨酸脱羧酶（ODC，ornithine decarboxylase）催化鸟氨酸脱羧形成[4-6]。腐胺在腐胺 N-甲基转移酶（PMT，putrescine-N-methyltransferase）作用下获得由 S-腺苷蛋氨酸（SAM，S-adenosyl-L-methionine）提供的甲基形成 N-甲基腐胺[7-9]，这是一个依赖S-腺苷蛋氨酸合成酶（SAMS，S-adenosylmethionine synthase）活性的反应。N-甲基腐胺在N-甲基腐胺氧化酶（MPO，N-methylputrescine oxidase）催化下形成4-甲氨基丁醚[10]，并通过自身环化形成N-甲基-△1-吡咯啉阳离子，随后与提供吡啶环部分的烟酸衍生物发生缩合反应形成烟碱[11]。

烟碱吡啶环部分由烟酸提供，其前体是由天冬氨酸合成的喹啉酸[12]。喹啉酸在喹啉酸磷酸核糖转移酶（QPRT，quinolinate phosphoribosyltransferase）催化下形成烟酰胺腺嘌呤二核苷酸（NAD），然后经由吡啶核苷酸循环途径生成烟酸[7,13]。

近期有关烟碱吡咯烷环部分和吡啶环部分缩合反应的研究表明，NADPH依赖性还原酶的PIP 家族（包括松脂醇还原酶、异黄酮还原酶和苯基香豆满苄基醚还原酶）成员类异黄酮还原酶基因A622及其同源基因参与了这一过程[14-15]，小檗碱桥接酶（Berberine bridge enzyme）家族成员BBL基因也参与了这一反应[16]。

2 烟碱转运相关基因

烟碱在烟草根尖细胞合成后[2,17]，经木质部运输到烟叶的叶肉细胞并储存于液泡内，推测应有大量蛋白参与了烟碱的转运过程。近期研究已从烟草中分离鉴定了若干烟碱转运蛋白基因，主要有定位于液泡膜的烟碱转运蛋白基因及定位于质膜的烟碱转运蛋白基因。

目前发现的定位于液泡膜的烟碱转运蛋白基因主要是多药与毒性化合物外排家族（MATE，multidrug and toxic compound extrusion）基因。MATE是一类新型二级转运蛋白基因家族，在植物中有多个成员[18]。2009年，Morita等从烟草中分离到一个MATE家族的转运蛋白基因，该基因受茉莉酸诱导表达，被命名为茉莉酸诱导烟碱转运蛋白基因JAT1[19]。JAT1蛋白定位于烟草叶片细胞的液泡膜上，可以转运烟碱及假木贼碱[19]。同时，另外两个烟碱转运蛋白基因 MATE1 和MATE2也在烟草中得到分离，MATE1和MATE2蛋白也定位于烟草叶片细胞的液泡膜上[20]。

最近分离鉴定的烟草尼古丁吸收透性酶（NUP1，nicotine uptake permease 1）基因编码另一类具有烟碱转运功能的蛋白，与定位于液泡膜上的MATE蛋白不同，NUP1蛋白定位于质膜上，负责将胞质外体的烟碱转运到胞质内[21]。此外，NUP1与MATE蛋白的烟碱转运功能也有差别[21]。

3 烟碱转化相关基因

烟碱是烟草特有亚硝胺NNK[4-(N-甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮]和NNN（N'–亚硝基降烟碱）的前体[22]。烟草中烟碱向降烟碱的转化由细胞色素P450基因家族中CYP82E亚族成员编码的烟碱-N-去甲基化酶（NND，nicotine N-demethylase）催化[23]。降烟碱是一种有害物质，是致癌物N'–亚硝基降烟碱（NNN）的主要前体[24-25]。目前已有多个催化烟碱向降烟碱转化的基因得到分离和鉴定，如：CYP82E5v2[23]、CYP82E2[26]、CYP82E10[27]和CYP82E4[28]等。在这些烟碱-N-去甲基化酶基因中，CYP82E5v2在绿色叶片中表达量较高，CYP82E4则主要在衰老叶片中表达[23]。近期还发现一个亚甲基四氢叶酸还原酶 MTHFR1（Methylenetetrahydrofolate reductase 1）可以通过调控CYP82E4基因的表达水平而影响烟碱向降烟碱的转化[29]。

4 烟碱代谢相关调控基因

烟碱合成受到多种因子调控，已知参与烟碱代谢调控的植物激素主要有茉莉酸、生长素及乙烯，其中生长素和乙烯是烟碱合成的负调控因子[4,30-33]。近期的烟碱代谢调控研究主要集中于茉莉酸信号途径调控因子。茉莉酸（JA，jasmonate）是重要的植物次生代谢调节因子，烟草中大部分烟碱合成功能基因都受到茉莉素信号途径调控[4,30,32,34-36]。

茉莉酸受体是由COI1、负调控因子JAZ（Jasmonate ZIM-Domain）蛋白和肌醇戊基磷酸分子组成的复合体[37-38]。有茉莉酸存在时，茉莉酸衍生物JA-Ile与茉莉酸受体相结合导致负调控因子JAZ蛋白的泛素化降解，从而释放下游转录激活因子并激活植物的茉莉酸应答[39]。烟草的茉莉酸途径调控因子COI1和JAZ蛋白都已被证明是烟碱合成调控因子[35]。近期研究还鉴定了一些调控烟碱合成的转录因子，如 ERF转录因子家族成员JAP1、ERF32及ORC1的同源基因等[40-41]，bHLH转录因子家族成员bHLH1/2和MYC2等[42-43]，这些ERF转录因子和bHLH转录因子还可以通过彼此间的相互调控影响烟碱代谢过程[44-45]。

前期对烟碱合成基因PMT启动子中一段由G-box，AT-rich和GCC-box-like元件构成的茉莉酸应答片段的研究证明这三个元件都是茉莉酸应答的必需元件[32]，已鉴定的ERF和bHLH烟碱合成调控因子可结合其中的G-box和GCC-box-like元件[41,43]，但AT-rich元件的结合蛋白还未发现，现有研究也无法解释PMT基因的茉莉酸应答机理。因此，烟碱合成代谢相关调控因子基因的分离和鉴定仍是一个重要研究方向。

5 问题与展望

虽然烟草的烟碱代谢途径已基本清楚，且大部分代谢步骤都有一些功能基因得到分离鉴定，但是这些功能基因在烟草中的同源基因远未得到完全发掘，特别是普通烟草为多倍体植物，每个基因都有大约5个同源基因，因此，烟碱代谢功能基因的研究还有大量工作需要开展。近来，烟碱代谢功能基因的调控机理研究已经成为烟碱代谢调控研究的热点问题，分离和鉴定烟碱代谢功能基因的调控因子将是未来一段时期的主要研究内容。

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