Analysis of cytokinin metabolism in ipt transgenic tobacco by liquid chromatography tandem mass spectrometry
Analysis of cytokinin metabolism in ipt transgenic tobacco by liquid chromatography tandem mass spectrometry
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DOI：
10.1104/pp.112.1.141
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发表时间：
1996-09-01
期刊：
影响因子：
7.4
通讯作者：
VanOnckelen, H
中科院分区：
文献类型：
--
作者：
Redig, P;Schmulling, T;VanOnckelen, H
以1，5-二磷酸核酮糖羧化酶小亚基启动子-异戊烯基转移酶基因（Pssu-ipt）转化烟草（Nicotiana tabacum L.）在6周的继代培养期间，使用电喷雾-液相色谱-串联质谱对愈伤组织进行定量。ipt基因在四环素诱导型启动子的控制下表达，以更详细地研究细胞分裂素的积累和代谢。两种表达系统中ipt的激活导致主要产生玉米蛋白型细胞分裂素。未观察到异戊烯基腺嘌呤或异戊烯基腺苷蓄积。在Pssu-ipt转化的愈伤组织，以及在四环素诱导ipt叶，代谢失活发生通过O-葡萄糖苷共轭。未观察到细胞分裂素N-葡萄糖苷水平显著升高。在这两个系统中观察到侧链还原为dihydrozeatin型细胞分裂素。内源性细胞分裂素的水平随时间变化，并受到稳态调节机制。用[H-3]异戊烯腺嘌呤和[H-3]异戊烯腺苷饲养ipt转基因愈伤组织，主要产生标记的腺嘌呤类化合物，它们是细胞分裂素氧化酶活性的降解产物。在玉米素核苷中观察到放射性掺入，尽管程度要小得多。
The endogenous levels of the major, naturally occurring cytokinins in Pisum sativum ribulose-1,5-bisphosphate carboxylase small subunit promoter-isopentenyl transferase gene (Pssu-ipt)transformed tobacco (Nicotiana tabacum L.) callus were quantified using electrospray-liquid chromatography-tandem mass spectrometry during a 6-week subcultivation period. An ipt gene was expressed under control of a tetracycline-inducible promoter for a more detailed study of cytokinin accumulation and metabolism. Activation of the ipt in both expression systems resulted in the production of mainly zeatin-type cytokinins. No accumulation of isopentenyladenine or isopentenyladenosine was observed. In Pssu-ipt-transformed calli, as well as in the tetracycline-inducible ipt leaves, metabolic inactivation occurred through O-glucoside conjugation. No significant elevation of cytokinin N-glucosides levels was observed. Side-chain reduction to dihydrozeatin-type cytokinins was observed in both systems. The levels of the endogenous cytokinins varied in time and were subject to homeostatic regulatory mechanisms. Feeding experiments of ipt transgenic callus with [H-3]isopentenyladenine and [H-3]isopentenyladenosine mainly led to labeled adenine-like compounds, which are degradation products from cytokinin-oxidase activity. Incorporation of radioactivity in zeatin riboside was observed, although to a much lesser extent.