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。在分子水平上研究果实成熟和软化的机理，将直接指导转基因植物的发展，使其具有更长的自我寿命。在这项研究中，我们克隆了编码纤维素酶和聚半乳糖醛酸酶的基因，这两个基因分别被认为可以修饰纤维素和果胶物质。在甜瓜果实中获得了6个编码纤维素酶的不同克隆。表达分析表明，这些基因可分为两组。第一类在幼果和膨大果中表达，表明在细胞生长中起生理作用，另一类则组成性表达，包含膜跨越结构域。令人惊讶的是，在正在成熟的果实中特异性表达的纤维素酶基因并没有从甜瓜果实中获得。在研究过程中，美国研究人员报道了甜瓜多半乳糖醛酸酶基因。因此，我们从与甜瓜同属黄瓜科的黄瓜和经历了密集生长的柿子中克隆了多乳糖醛酸酶基因。在果实成熟过程中迅速软化。结果表明，单长度聚半乳糖醛酸酶。从黄瓜果实中获得基因。表达分析表明，果实软化过程、水分胁迫和外源乙烯诱导了mRNA的积累。另一方面，强乙烯抑制剂1-MCP预处理可抑制其表达。结果表明，多半乳糖醛酸酶基因与胁迫-乙烯诱导果实软化有关。

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.