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