張洪博

（西南大學(xué)農(nóng)學(xué)與生物科技學(xué)院，重慶 400715）

煙堿（nicotine），即尼古丁，是煙草生物堿（包括煙堿、降煙堿、新煙草堿和假木賊堿等）的一種，約占煙草生物堿總含量的90%～95%[1]。煙葉煙堿含量占葉片干重的0.6%～3.0%，是煙草和卷煙質(zhì)量的一項(xiàng)重要指標(biāo)。對煙堿代謝的分子遺傳學(xué)研究可以揭示煙堿代謝累積的分子機(jī)制，并為煙堿含量及煙堿成分調(diào)節(jié)相關(guān)的育種工作提供理論基礎(chǔ)。近年來，有關(guān)煙堿合成、轉(zhuǎn)運(yùn)及轉(zhuǎn)化的一些重要基因已被陸續(xù)克隆，對煙堿合成代謝機(jī)理研究和煙草遺傳育種工作產(chǎn)生了重要推動作用。

1 煙堿合成相關(guān)基因

煙堿分子由一個吡咯烷環(huán)和一個吡啶環(huán)構(gòu)成，在煙草根部合成，通過木質(zhì)部向地上部運(yùn)輸，在煙草植株的葉片中含量最高，莖部含量最低[2-3]。

煙堿吡咯烷環(huán)由氮代謝中形成的腐胺合成。腐胺可通過精氨酸脫羧酶（ADC，arginine decarboxylase）催化精氨酸脫羧形成，或由鳥氨酸脫羧酶（ODC，ornithine decarboxylase）催化鳥氨酸脫羧形成[4-6]。腐胺在腐胺 N-甲基轉(zhuǎn)移酶（PMT，putrescine-N-methyltransferase）作用下獲得由 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]，這是一個依賴S-腺苷蛋氨酸合成酶（SAMS，S-adenosylmethionine synthase）活性的反應(yīng)。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]，并通過自身環(huán)化形成N-甲基-△1-吡咯啉陽離子，隨后與提供吡啶環(huán)部分的煙酸衍生物發(fā)生縮合反應(yīng)形成煙堿[11]。

煙堿吡啶環(huán)部分由煙酸提供，其前體是由天冬氨酸合成的喹啉酸[12]。喹啉酸在喹啉酸磷酸核糖轉(zhuǎn)移酶（QPRT，quinolinate phosphoribosyltransferase）催化下形成煙酰胺腺嘌呤二核苷酸（NAD），然后經(jīng)由吡啶核苷酸循環(huán)途徑生成煙酸[7,13]。

近期有關(guān)煙堿吡咯烷環(huán)部分和吡啶環(huán)部分縮合反應(yīng)的研究表明，NADPH依賴性還原酶的PIP 家族（包括松脂醇還原酶、異黃酮還原酶和苯基香豆?jié)M芐基醚還原酶）成員類異黃酮還原酶基因A622及其同源基因參與了這一過程[14-15]，小檗堿橋接酶（Berberine bridge enzyme）家族成員BBL基因也參與了這一反應(yīng)[16]。

2 煙堿轉(zhuǎn)運(yùn)相關(guān)基因

煙堿在煙草根尖細(xì)胞合成后[2,17]，經(jīng)木質(zhì)部運(yùn)輸?shù)綗熑~的葉肉細(xì)胞并儲存于液泡內(nèi)，推測應(yīng)有大量蛋白參與了煙堿的轉(zhuǎn)運(yùn)過程。近期研究已從煙草中分離鑒定了若干煙堿轉(zhuǎn)運(yùn)蛋白基因，主要有定位于液泡膜的煙堿轉(zhuǎn)運(yùn)蛋白基因及定位于質(zhì)膜的煙堿轉(zhuǎn)運(yùn)蛋白基因。

目前發(fā)現(xiàn)的定位于液泡膜的煙堿轉(zhuǎn)運(yùn)蛋白基因主要是多藥與毒性化合物外排家族（MATE，multidrug and toxic compound extrusion）基因。MATE是一類新型二級轉(zhuǎn)運(yùn)蛋白基因家族，在植物中有多個成員[18]。2009年，Morita等從煙草中分離到一個MATE家族的轉(zhuǎn)運(yùn)蛋白基因，該基因受茉莉酸誘導(dǎo)表達(dá)，被命名為茉莉酸誘導(dǎo)煙堿轉(zhuǎn)運(yùn)蛋白基因JAT1[19]。JAT1蛋白定位于煙草葉片細(xì)胞的液泡膜上，可以轉(zhuǎn)運(yùn)煙堿及假木賊堿[19]。同時，另外兩個煙堿轉(zhuǎn)運(yùn)蛋白基因 MATE1 和MATE2也在煙草中得到分離，MATE1和MATE2蛋白也定位于煙草葉片細(xì)胞的液泡膜上[20]。

最近分離鑒定的煙草尼古丁吸收透性酶（NUP1，nicotine uptake permease 1）基因編碼另一類具有煙堿轉(zhuǎn)運(yùn)功能的蛋白，與定位于液泡膜上的MATE蛋白不同，NUP1蛋白定位于質(zhì)膜上，負(fù)責(zé)將胞質(zhì)外體的煙堿轉(zhuǎn)運(yùn)到胞質(zhì)內(nèi)[21]。此外，NUP1與MATE蛋白的煙堿轉(zhuǎn)運(yùn)功能也有差別[21]。

3 煙堿轉(zhuǎn)化相關(guān)基因

煙堿是煙草特有亞硝胺NNK[4-(N-甲基亞硝胺基)-1-(3-吡啶基)-1-丁酮]和NNN（N'–亞硝基降煙堿）的前體[22]。煙草中煙堿向降煙堿的轉(zhuǎn)化由細(xì)胞色素P450基因家族中CYP82E亞族成員編碼的煙堿-N-去甲基化酶（NND，nicotine N-demethylase）催化[23]。降煙堿是一種有害物質(zhì)，是致癌物N'–亞硝基降煙堿（NNN）的主要前體[24-25]。目前已有多個催化煙堿向降煙堿轉(zhuǎn)化的基因得到分離和鑒定，如：CYP82E5v2[23]、CYP82E2[26]、CYP82E10[27]和CYP82E4[28]等。在這些煙堿-N-去甲基化酶基因中，CYP82E5v2在綠色葉片中表達(dá)量較高，CYP82E4則主要在衰老葉片中表達(dá)[23]。近期還發(fā)現(xiàn)一個亞甲基四氫葉酸還原酶 MTHFR1（Methylenetetrahydrofolate reductase 1）可以通過調(diào)控CYP82E4基因的表達(dá)水平而影響煙堿向降煙堿的轉(zhuǎn)化[29]。

4 煙堿代謝相關(guān)調(diào)控基因

煙堿合成受到多種因子調(diào)控，已知參與煙堿代謝調(diào)控的植物激素主要有茉莉酸、生長素及乙烯，其中生長素和乙烯是煙堿合成的負(fù)調(diào)控因子[4,30-33]。近期的煙堿代謝調(diào)控研究主要集中于茉莉酸信號途徑調(diào)控因子。茉莉酸（JA，jasmonate）是重要的植物次生代謝調(diào)節(jié)因子，煙草中大部分煙堿合成功能基因都受到茉莉素信號途徑調(diào)控[4,30,32,34-36]。

茉莉酸受體是由COI1、負(fù)調(diào)控因子JAZ（Jasmonate ZIM-Domain）蛋白和肌醇戊基磷酸分子組成的復(fù)合體[37-38]。有茉莉酸存在時，茉莉酸衍生物JA-Ile與茉莉酸受體相結(jié)合導(dǎo)致負(fù)調(diào)控因子JAZ蛋白的泛素化降解，從而釋放下游轉(zhuǎn)錄激活因子并激活植物的茉莉酸應(yīng)答[39]。煙草的茉莉酸途徑調(diào)控因子COI1和JAZ蛋白都已被證明是煙堿合成調(diào)控因子[35]。近期研究還鑒定了一些調(diào)控?zé)焿A合成的轉(zhuǎn)錄因子，如 ERF轉(zhuǎn)錄因子家族成員JAP1、ERF32及ORC1的同源基因等[40-41]，bHLH轉(zhuǎn)錄因子家族成員bHLH1/2和MYC2等[42-43]，這些ERF轉(zhuǎn)錄因子和bHLH轉(zhuǎn)錄因子還可以通過彼此間的相互調(diào)控影響煙堿代謝過程[44-45]。

前期對煙堿合成基因PMT啟動子中一段由G-box，AT-rich和GCC-box-like元件構(gòu)成的茉莉酸應(yīng)答片段的研究證明這三個元件都是茉莉酸應(yīng)答的必需元件[32]，已鑒定的ERF和bHLH煙堿合成調(diào)控因子可結(jié)合其中的G-box和GCC-box-like元件[41,43]，但AT-rich元件的結(jié)合蛋白還未發(fā)現(xiàn)，現(xiàn)有研究也無法解釋PMT基因的茉莉酸應(yīng)答機(jī)理。因此，煙堿合成代謝相關(guān)調(diào)控因子基因的分離和鑒定仍是一個重要研究方向。

5 問題與展望

雖然煙草的煙堿代謝途徑已基本清楚，且大部分代謝步驟都有一些功能基因得到分離鑒定，但是這些功能基因在煙草中的同源基因遠(yuǎn)未得到完全發(fā)掘，特別是普通煙草為多倍體植物，每個基因都有大約5個同源基因，因此，煙堿代謝功能基因的研究還有大量工作需要開展。近來，煙堿代謝功能基因的調(diào)控機(jī)理研究已經(jīng)成為煙堿代謝調(diào)控研究的熱點(diǎn)問題，分離和鑒定煙堿代謝功能基因的調(diào)控因子將是未來一段時期的主要研究內(nèi)容。

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