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

细胞壁是决定植物物理结构的主要因素，也是制约植物贮藏、运输和销售的主要制约因素。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，细胞壁分解的关键因素尚未确定。“La France”欧洲梨果和甜瓜果实在乙烯依赖的成熟过程中经历了戏剧性的软化，而中国梨尽管在成熟过程中产生了大量的乙烯，但却没有表现出软化。为了广泛、全面地筛选果实软化的影响因素，除了对多半乳糖醛酸酶、α-阿拉伯糖葡糖苷酶和β-半乳糖苷酶进行基因表达分析外，还采用了二维凝胶电泳图谱和细胞壁蛋白的YSST分析。从成熟的‘La France’梨中分离出200个蛋白质斑点的积累水平。用1-MCP（一种强乙烯抑制剂）处理该品种的软化梨，可延缓果实软化。通过对处理和未处理果实的比较，选择了3个蛋白质斑点作为果实软化的关键因子候选，因为它们的积累具有特定的软化模式。在YSST分析中，分离到170个编码分泌蛋白的克隆（70个contigs和100个singleton），其中包括以前未报道的克隆。经macroarray分析，28个无性系在梨果实发育和成熟过程中表达上调或下调。通过对‘La France’和‘Yali’梨果实的比较，从28个无性系中筛选出11个潜在的细胞壁修饰蛋白作为果实软化关键因子的候选。

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.