Gene clone, expression and enzyme activity assay of a cytosolic malate dehydrogenase from apple fruits

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 (GenBank accession No. DQ221207). It contained a 996-bp ORF and its sequence analysis shows 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. A roughly 37-kDa 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 remarkable difference between the high- and low-acid apple germplasm.

Keywords

apple / cytosolic malate dehydrogenase / gene clone / prokaryotic expression / enzyme activity