园艺学报 ›› 2008, Vol. 35 ›› Issue (2): 181-188.

• 果树 • 上一篇    下一篇


姚玉新1;郝玉金1;李 明1; 庞明利1;刘 志2 ;翟 衡1*   

  1. 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)
  • 收稿日期:2007-06-20 修回日期:2007-11-22 出版日期:2008-02-25 发布日期:2008-02-25
  • 通讯作者: 翟 衡

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

YAO Yu-xin 1,HAO Yu-jin1, LI Ming1,PANG Ming-li 1,LIU Zhi2 ,and ZHAI Heng1*

  1. (1 College of Horticultural Science and Engineering, Shandong Agricultural University, State Key Laboratory of Crop Biology, Tai'an, Shandon 271018, China; 2 Liaoning Institute of Fruit Tree Science, Xiongyue, Liaoning 115214, China)
  • Received:2007-06-20 Revised:2007-11-22 Online:2008-02-25 Published:2008-02-25
  • Contact: ZHAI Heng

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(Genebank登录号为DQ221207),该序列含有一个996 bp的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37 kD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。

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

Abstract: 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 malic acid synthesis in the cytosol, which has not been extensively characterized in plants. In this study, a full-length cDNA of cyMDH was isolated from apple fruits with RT-PCR as well as 3′ and 5′ rapid amplification of cDNA ends, and designated as Mal-cyMDH (GeneBank accession No. DQ221207). It contained a 996-bp ORF and sequence analysis showed a high similarity to other plant cyMDHs. Phylogenetic 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.

Key words: Apple, cytosolic malate dehydrogenase, gene cloning, prokaryotic expression, enzyme activity