一类依赖于NAD的玉米胞质型苹果酸脱氢酶基因的克隆及其序列分析

苹果酸脱氢酶普遍存在于各种生物中,它负责催化草酰乙酸和苹果酸之间的相互转换.根据其辅酶的特异性和在细胞内的分布及其生理功能的不同,苹果酸脱氢酶在高等植物中可以区分出不同的类型,依赖于NAD的细胞质型苹果酸脱氢酶是其中研究较少的一类.根据已发表的其他高等植物的依赖于NAD的胞质型苹果酸脱氢酶基因的保守序列,运用SMART RACE RT-PCR技术,从玉米叶片中分离了cyMDH 的1 264 bp全长cDNA序列,通过生物信息学分析发现,该序列含有一个999 bp的完整的开放阅读框,其共编码332个氨基酸(GenBank登陆号 EU625276).序列联配与树状分析结果表明,该玉米cyMDH 序列与多个物种的cyMDH 基因具有高度的同源性.组织特异性表达分析显示MDH基因在玉米叶片中表达量最高,在茎、根中亦有低水平表达.本研究将为更深入的研究玉米cyMDH 基因的分子调控机理奠定基础.

Cloning and Sequence Analysis of the NAD-dependent Cytosolic Malate Dehydrogenase Gene from Zea Mays

Malate dehydrogenase(MDH) which catalyzes the reversible reaction from oxaloacetate to malate ubiquitously exists in nature. Higher plants contain multiple forms of MDHs that differ in co-enzyme specificity, subcellular localization and physiological function. The NAD-dependent cytosolic MDH (cyMDH ) is one class of MDH that is still seldom investigated. On the basis of the conserved amino acid residues in the published cytosolic malate dehydrogenase protein sequences from other higher plant species, a full-length cDNA was amplified by SMART RACE RT-PCR from the total RNA of maize leaves. Bioinformatics analysis of the cDNA sequence indicates that the 264 bp full length of the target clone includes a 70 bp 5'-UTR, an ORF of 999 bp, and a 195 bp 3'-UTR (GenBank accession EU625276). This cDNA sequence codes 332 amino acids residues with a predicted molecular mass of 35.6168 kD and an isoelectric point (pI) of 5.4. The deduced maize amino acids share high sequence homology with cyMDH from other species, such as O.sative, M.crystallinum, and G.max. Analysis of semiquantitive RT-PCR shows that the expression of cyMDH is higher in maize leaves than that in roots and stems. All of these results will provide a theoretical basis for further investigation of the cyMDH gene in molecular regulation mechanism.