Identification of key factor fur cell wall disassembly in fruit by proteome analysis and transgenic technology

细胞壁是植物物理结构的主要因素，也是限制植物贮藏、运输和商品化的主要限制因素。以往的研究表明，植物激素乙烯对果实成熟和软化的重要性。然而，细胞壁解体的关键因素尚未确定。"法国“欧洲梨果实和甜瓜果实在成熟过程中以乙烯依赖的方式发生急剧软化，而中国梨尽管在成熟过程中产生大量乙烯，但没有表现出软化。为了大范围、全面地筛选影响果实软化的因素，本研究除对多聚半乳糖醛酸酶、α-阿拉伯呋喃糖苷酶和β-半乳糖苷酶基因表达进行分析外，还采用了双向凝胶电泳和细胞壁蛋白YSST分析等技术。在成熟的'La France'梨中分离出200个蛋白质点的积累水平。用乙烯抑制剂1-MCP处理该品种软化梨，可延缓果实软化。通过对处理前后果实的比较，筛选出3个蛋白质点作为影响果实软化的关键因子，经YSST分析，共获得170个分泌蛋白克隆（70个contigs和100个singletons），其中包括一些未见报道的克隆。根据基因芯片分析，有28个克隆在梨果实发育和成熟过程中表达上调或下调。通过对28个无性系果实的比较，筛选出11个新的细胞壁修饰蛋白作为果实软化的关键因子。

Cell wall is main factor for physical structure of plant and the main limiting factors limiting their storage, transport and marketability. Previous studies indicated importance of plant hormone ethylene for fruit ripening and softening. However, key factor (s) in cell wall disassembly have not been identified. 'La France' European pear fruit and melon fruit undergo dramatic softening during ripening in an ethylene-dependent manner, whereas Chinese pear does not exhibit softening in spite of its massive ethylene production during ripening. To broad-scale and comprehensive screening for factors involved in fruit softening, in addition to gene expression analysis of polygalacturonase, α-arabinofuranosidase and β-galactosidase, the two-dimensional gel electrophoresis profiles and YSST analysis of cell wall proteins were employed. The accumulation levels of 200 protein spots were isolated in the ripening 'La France' pear. When softening pears of this variety were treated with 1-MCP, a strong ethylene inhibitor, fruit softening was retarded. On the comparison between treated and non-treated fruit, 3 protein spots were selected as candidates for key factors of fruit softening, since their accumulation showed specific pattern for softening.On YSST analysis, 170 clones (70 contigs and 100 singletons) encoding secreted proteins including clones which have not been previously reported were isolated. Based on the macroarray analysis, 28 clones showed elevated or down-regulated expression with pear fruit development and ripening. On comparison in 'La France' and the non-softening 'Yali' pear fruit, 11 potential new cell wall modifying proteins among 28 clones were selected for candidates for key factor of fruit softening.