Characterization of a cytosolic malate dehydrogenase cDNA which encodes an isozyme toward oxaloacetate reduction in wheat
Characterization of a cytosolic malate dehydrogenase cDNA which encodes an isozyme toward oxaloacetate reduction in wheat
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DOI：
10.1016/j.biochi.2004.07.011
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发表时间：
2004-08-01
期刊：
影响因子：
3.9
通讯作者：
Ma, QH
中科院分区：
文献类型：
--
作者：
Ding, Y;Ma, QH
苹果酸脱氢酶(MDH)是自然界中普遍存在的一种催化草酰乙酸酯和苹果酸相互转化的酶。高等植物含有多种形式的MDH，它们在辅酶专一性、亚细胞定位和生理功能上存在差异。胞液NAD依赖的MDH(CyMDH)是一类在植物中尚未被广泛研究的MDH。本文报道了用RT-PCR和文库筛选的方法从小麦中克隆到一个新的基因，命名为TaMDH。序列分析表明，TaMDH与其他植物的CyMDH有很高的相似性。免疫学分析证实，TaMDH编码胞内NAD依赖的MDH。通过分子模拟对TaMDH的二级结构和三维结构进行了分析。DNA凝胶印迹分析表明，TaMDH基因在小麦基因组中以双拷贝形式存在。RNA和蛋白质凝胶印迹杂交表明，TaMDH在小麦营养组织中均有结构性表达，但根中的表达略低于叶和茎中的表达。计算机分析表明，在多种胁迫条件下，TaMDH也在不同的生殖组织和组织中表达。细菌表达和纯化蛋白的动力学分析证实，TaMDH催化了苹果酸合成反应，这与其他cyMDH一致。进化分析表明，这类基因是从一个非常古老的基因进化而来的。CyMDH代表MDH的祖先形式，在植物、动物和细菌中高度保守。这意味着cyMDH是看家基因，可能在植物新陈代谢中具有非常重要的功能。(C)2004年爱思唯尔公司。版权所有。
Malate dehydrogenase (MDH), which is ubiquitous in nature, catalyzes the interconversion of oxaloacetate and malate. Higher plants contain multiple forms of MDH that differ in co-enzyme specificity, subcellular localization and physiological function. Cytosolic NAD-dependent MDH (cyMDH) is one class of MDH that has not been extensively characterized in plants. Here we report the cloning of a cDNA from wheat by RT-PCR and cDNA library screening, which is designated as TaMDH. Sequence analysis indicated that TaMDH exhibits a highly similarity to other plant cyMDHs. Immunological analysis confirmed that TaMDH encoded a cytosolic NAD-dependent MDH. The secondary and three-dimensional structures of TaMDH were analyzed by molecular modeling. DNA gel-blot analyses demonstrated that TaMDH gene exists as two copies in the wheat genome. RNA and protein gel-blot hybridization indicated that both TaMDH mRNA and protein were constitutively expressed in vegetative tissues of wheat, with slightly lower levels in roots than in leaves and stems. In silico analysis indicated that TaMDH was also expressed in various reproductive tissues and tissues under many different stress conditions. Kinetic analysis of bacterially expressed and purified protein confirmed that TaMDH catalyzed a reaction driven towards malate synthesis, which is consistent with other cyMDHs. Evolutionary analysis showed that this class of genes evolved from a very ancestral gene. The cyMDH represents an ancestral form of MDH, which is highly conserved in plants, animals and bacteria. This implies that cyMDHs are housekeeping genes and may have very essential functions in plant metabolism. (C) 2004 Elsevier SAS. All rights reserved.