Isolation And Expression Analysis Of Alcohol Dehydrogenase (ADH) Gene From Banana (Musa Ssp.)

Banana is one of the most important fruit in the world, which is ranked as the world’s fourth most important food crop. Some adversity, such as waterlogging and low temperature and so on, affect banana seedling growth and fruit production during banana growth and development. Banana fruit is a typical climacteric fruit, which concerns with banana fruit quality and shelf life.Banana seedling growth and fruit ripening have a direct impact on the development of the banana industry. Alcohol dehydrogenase (alcohol dehydrogenase, ADH, EC 1.1.1.1.) widely distributed in various organs and tissues of plants. ADH was found to play important roles in plant stress tolerance, growth, development, fruit ripening, senescence and aromatic biosynthesis. So, investigating the relationship between ADH and banana seedling growth and fruit ripening is an important issue, which will offer us more resistant banana seedlings and improve post-harvest preservation and ripening technology,In order to gain a better understanding of the mechanism about banana postharvest ripening, we isolate differentially cDNAs at the early stage of banana ripening by suppression subtractive hybridization. A total of 289 clones in the SSH library are sequenced. BlastX showed that a 500bp fragment was very similar with other plants ADH gene. We designed degenerate primers to obtain two full-length of ADHs named MaADHl and MaADH2. The full length of MaADH2 is 1140 bp with a complete open reading frame, encoding 379 amino acids that shared high identities of 92%,92% and 91% with that from Arabidopsis thaliana, Ricinus communis, Oryza sativa respectively. Conserved domain analysis showed that this cDNA contain two conserved sites, ADH_N superfamily and NADB_ROssmann superfamily, which are typical ADH gene domain match. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that MaADH2 was expressed in root,stem,leave,flower and fruit.The expression level was higher in roots than other organs. Therefore, MaADH2 may play a certain role in different physiological processes.In order to investigate the relation between MaADH2 and banana ripening, we measured the ethylene production and analysed the expression level of MaADH2 at different stages with the treatment of ethylene and 1-MCP. The results showed that ethylene induced the banana fruit ripening but did not induce MaADH2 expression.1-MCP inhibited banana fruit ripening. However, the expression of MaADH2 did not decrease when the endogenous ethylene was inhibited.In order to study the relation between MaADH2 and exogenous ABA in banana fruit ripening, we analysed the expression level of MaADH2 at different stages with the treatment of ABA. The results showed that ABA delayed the expression level of MaADH2 to reach a maximum value.To research the relationship between MaADH2 and under-stress banana seedlings, we used RT-PCR to analyse the expression level of MaADH2 in leaves at injury stress, salt stress, low temperature stress.We used real-time QPCR to analyse the expression level of MaADH2 in leaves and roots at drought stress and waterlogging stress. Results showed that the injury stress negatively regulated MaADH2expression. Salt stress up-regulated it.Low temperature induced it.Mild drought stress induced the expression level of MaADH2, but it is down with drought increasing. Waterlogging stress induced the expression level of MaADH2 in roots, but it changed little in leaves. We measured MaADH2 and MaPDC activity in normal roots and waterlogging stress roots and found that MaADH2 and MaPDC activities peaked at 24 hours, then declined under waterlogging stress in banana roots.According to these results, the MaADH2 gene was not related to fruit ripening but closely related to various of stress.