Chintapakorn Yupynn, Hamill John D
School of Biological Sciences, Monash University, P.O. Box 18, Melbourne, Victoria 3800, Australia.
Plant Mol Biol. 2003 Sep;53(1-2):87-105. doi: 10.1023/B:PLAN.0000009268.45851.95.
Nicotiana tabacum L. produces a number of pyridine alkaloids, with nicotine representing the major component and anatabine comprising most of the remainder of the alkaloid fraction. An antisense approach was used here to down-regulate activity of the important enzyme putrescine N-methyltransferase (PMT) in transformed roots of this species to determine effects upon alkaloid metabolism. Transformed root lines were produced that contained markedly reduced PMT activity, with a concomitant reduction in nicotine content compared to controls. No negative effects upon growth were observed. Several antisense-PMT transformed root lines, and also leaf tissues of regenerated transformed plants, showed a substantial increase in anatabine content relative to controls. Northern hybridization experiments indicated that the antisense-PMT manipulation had little or no effect upon the transcript levels of other genes encoding enzymes involved in alkaloid metabolism, including quinolinate acid phosphoribosyltransferase (QPT). The latter enzyme plays a key role in regulating the synthesis of nicotinic acid which supplies the pyridine ring necessary for both nicotine and anatabine synthesis. We suggest that elevated anatabine levels in antisense-PMT lines are a direct consequence of a relative oversupply of nicotinic acid which, in the absence of adequate levels of 1-methyl-delta(1)-pyrrolinium cation (the ultimate product of PMT activity), is used to synthesise anatabine directly. As is discussed, no naturally occurring species or varieties of Nicotiana are known that typically produce high levels of anatabine in root or leaf tissues, meaning that the antisense PMT transgenics produced in this study have no natural counterpart. These experiments thus represent an example of metabolic engineering of plant pyridine metabolism, via antisense down-regulation of gene expression in a contributing pathway leading to secondary metabolite biosynthesis.
烟草（Nicotiana tabacum L.）能产生多种吡啶生物碱，其中尼古丁是主要成分，降烟碱则构成了生物碱部分的其余大部分。本研究采用反义技术下调该物种转化根中重要酶腐胺N -甲基转移酶（PMT）的活性，以确定其对生物碱代谢的影响。所产生的转化根系中PMT活性显著降低，与对照相比，尼古丁含量也随之减少。未观察到对生长有负面影响。几个反义PMT转化根系以及再生转化植株的叶片组织，与对照相比，降烟碱含量大幅增加。Northern杂交实验表明，反义PMT操作对参与生物碱代谢的其他编码酶基因的转录水平几乎没有影响，包括喹啉酸磷酸核糖基转移酶（QPT）。后一种酶在调节烟酸合成中起关键作用，烟酸为尼古丁和降烟碱合成提供所需的吡啶环。我们认为，反义PMT品系中降烟碱水平升高是烟酸相对供应过剩的直接结果，在缺乏足够水平的1 -甲基 - δ(1)-吡咯啉阳离子（PMT活性的最终产物）的情况下，烟酸被直接用于合成降烟碱。如所讨论的，尚无已知的烟草天然物种或变种在根或叶组织中通常产生高水平的降烟碱，这意味着本研究中产生的反义PMT转基因植株没有天然对应物。因此，这些实验代表了通过反义下调导致次生代谢物生物合成的相关途径中的基因表达来进行植物吡啶代谢代谢工程的一个实例。
