Molecular cloning of genes related to fruit softening and their expression in melon fruit

最近，植物的基因操作有了巨大的进步。从分子水平上研究果实成熟软化的机制，将直接导致转基因植物的发展，具有更长的自我生活。在这项研究中，我们克隆了编码纤维素酶和多聚半乳糖醛酸酶的基因，这两种酶分别被认为可以修饰纤维素和果胶物质。我们获得了6个编码甜瓜果实纤维素酶的趋异克隆。表达分析表明，这些基因分为两组。第一组在幼果和膨大果实中表达，表明在细胞生长中的生理作用，另一组组成型表达并含有跨膜结构域。令人惊讶的是，在正在经历成熟的果实中特异性表达的纤维素酶基因不是从甜瓜果实中获得的。在本研究过程中，美国研究人员报道了甜瓜的聚半乳糖醛酸酶基因，因此我们转而从与甜瓜同属黄瓜科的黄瓜和经过强化处理的柿果实中克隆聚半乳糖醛酸酶基因。在果实成熟过程中迅速软化。从结果来看，一个长度的多聚半乳糖醛酸酶。基因是从黄瓜果实中获得的。表达分析表明，mRNA的积累诱导果实软化的进展，水分胁迫或外源乙烯。另一方面，表达被抑制的预处理与1-MCP，乙烯作用的强抑制剂。结果表明，多聚半乳糖醛酸酶基因与乙烯胁迫诱导的果实软化有关。

Recently, the genetic manipulation of plants has tremendously advanced. The study of the mechanism of fruit ripening and softening at molecular level, will result directly to the development of transgenetic plants that have a longer self-life. In this study, we cloned genes encoding cellulase and polygalacturonase which have been known to modify cellulose and pectin substances respectively. We obtained six divergent clones which encode cellulase in melon fruit. The expression analysis indicated that those genes were divided into two groups. The first group is expressed in young and expanding fruit, suggesting a physiological role in cell growth, and other group is expressed constitutively and contains membrane spanning domain. Surprisingly, a cellulase gene expressed specifically in fruit that is undergoing ripening was not obtained from melon fruit. During the course of this study, polygalacutoronase gene of melon was reported by researchers in USA.We therefore turned to clone polygaracturonase genes from cucumber, that belongs to Cucumis family as same as melon, and from persimmon fruit that undergo an intensive and. rapid softening during fruit ripening. As for the results, one length polygalacturonase. gene was obtained from cucumber fruit. Expression analysis showed that the accumulation of the mRNA was induced with progress of fruit softening, by water stress or by exogenous ethylene. On the other hands the expression was inhibited by the pretreatment with 1-MCP, a strong inhibitor of ethylene action. The results suggest that the polygalacturonase gene is responsible for fruit softening induced by stress-ethylene.