苹果细胞质型苹果酸脱氢酶基因克隆、表达及酶活性分析  

Gene Cloning,Expression and Enzyme Activity Assay of a Cytosolic Malate Dehydrogenase from Apple Fruits

作　　者：姚玉新[1] 郝玉金[1] 李明[1] 庞明利[1] 刘志[2] 翟衡[1] 

机构地区：[1]山东农业大学园艺科学与工程学院,作物生物学国家重点实验室,山东泰安271018 [2]辽宁省果树科学研究所,辽宁熊岳115214

出　　处：《园艺学报》2008年第2期181-188,共8页Acta Horticulturae Sinica

摘　　要：根据亚细胞定位,苹果酸脱氢酶可分为5种类型,其中依赖于NAD的细胞质型苹果酸脱氢酶(cyMDH)在植物上的研究较少。从苹果果实中分离了cyMDH的全长cDNA序列,命名为Mal-cyMDH(GenBank登录号为DQ221207),该序列含有一个996bp的开放阅读框(ORF)。序列同源性分析表明Mal-cyMDH与其他物种cyMDH有较高的同源性,进化树分析表明几乎所有的cyMDH可以聚类在一个分支上,并且cyMDH可能是MDH的最原始种。原核表达得到一个37kD左右的融合蛋白,酶活测定表明该酶主要催化合成苹果酸。苹果果实中cyMDH酶活分析表明在高酸和低酸基因型中该酶的还原活性存在明显差异。Malate dehydrogenase （MDH） ubiquitously exists in animals, plants and microoganisms, and catalyzes the interconversion from oxaloacetate to malate. Cytosolic NAD-dependent MDH gene （cyMDH） encodes a key enzyme crucial for malie acid synthesis in the cytosol, which has not been extensively characterized in plants. In this study, a full-length eDNA of cyMDH was isolated from apple fruits with RT-PCR as well as 3′ and 5′ rapid amplification of eDNA ends, and designated as Mal-cyMDH （ GenBank accession No. DQ221207）. It contained a 996 bp ORF and sequence analysis showed a high similarity to other plant eyMDHs. Phylogenetie analysis indicated that almost all the cyMDHs could be clustered into the same group and it was likely to represent the original MDH. An about 37 kD fused protein was obtained by the recombinant prokaryotic expression and its enzyme activity assay showed that it mainly catalyzed oxaloacetate to malate. It was also discovered that the enzyme activity of cyMDH exhibited remarkably difference between the high- and low-acid apple germplasm.

关 键 词：苹果 细胞质型苹果酸脱氢酶 基因 克隆 原核表达 酶活性 

分 类 号：S661.1[农业科学—果树学]