Isolation And Expression Analysis Of Malate Dehydrogenase (MDH) Gene From Banana (Musa Spp.)

Malate dehydrogenase (MDH) ubiquitously existed in plants, animals and bacteriums with a high conservation, and catalyzed the interconversion of oxaloacetate and malate. Based upon coenzyme specificity, MDHs can be divided into NAD-dependent MDHs and NADP-dependent MDHs. MDH not only involved in many physiological activities, including the C4 cycle, respiration, fatty acid oxidation and TCA cycle, but also played an important role in seed germination, plant growth, pollen development, fruit development and pant stress tolerance. This study selected a fragment from a cDNA library of banana fruit constructed by workers. This gene was isolated from cDNA library by RACE. Bioinformatics analysis indicated that the full length of MaMDH cDNA was 1249bp with an open reading frame of 999bp, which encoded 332 amino acids, and had a high homology to malate dehydrogenase, so we designated it as MaMDH gene. MaMDH amino acid sequence contained NAD binding site, malate binding site and dimmer interface. Amino acid alignment showed a high similarity to other plant cyMDHs from Ricinus communis, Nicotiana tabacum, Prunus persica and Cicer arietinum, reaching to 90%. Real-time quantitative PCR analysis showed that MaMDH gene was constitutively expressed in roots, corms, leaves, flowers and fruits, with a higher expression level in flowers, fruits, roots and a lower expression in corms, leaves, suggesting that it may play a certain role in different physiological process.During the process of banana fruit postharvest ripening, we studied the correlations among MaMDH gene expression, enzyme activity and malic acid. mRNA level of MaMDH increased gradually with banana fruit postharvest ripening and then remaining relatively stable until the fruits ripened. In parallel, the enzyme activity of MDH increased gradually with fruit ripening, which was positively correlated with the expression level of MdMDH, but not with malate content, especially in the ripening fruit. Meanwhile, the total soluble sugar content also increased.In order to investigate the relation between MaMDH and ethylene biosynthesis, we measured the ethylene production and analysed the expression level of MaMDH at different stages with the treatment of ethylene and 1-MCP. When naturally ripened, the expression of MaMDH gene peaked at 14d after postharvest consistent with ethylene peak. When treated by ethylene, the expression of MaMDH gene peaked at 3d after postharvest similar to the ethylene production. But the expression of MaMDH gene was obviously inhibited by 1-MCP. These results demonstrated that ethylene induced expression of MaMDH gene.When treated with exogenous ABA and Fluridone respectively, expression of MaMDH gene was obviously inhibited in banana at different ripening stage.The transcriptional responses of MaMDH to different abiotic stresses were examined in banana seedlings. When treated by drought and wound stress, expression of MaMDH gene decreased. When treated by cold stress, expression of MaMDH gene was induced, peaked at 5°.Similarly, mRNA level of this gene was induced when treated by salt, ethrel and A13+ stress.We used Fusarium oxysporum f.sp.cubense race four (FOC4) infect roots of banana seedlings. The result indicated that MaMDH gene was induced, reaching peak when infected 4d.