【作者】 戴绍军；

【导师】 王台； 种康；

【作者基本信息】 中国科学院研究生院（植物研究所） ， 植物学， 2006， 博士

【摘要】 花粉是高度退化的两细胞或三细胞生物体。作为雄配子体,花粉储藏了父本的全部遗传信息,并通过与柱头识别、萌发和花粉管极性生长将精子送到雌性的胚囊中,完成双受精作用。由于花粉在植物有性生殖过程中的特殊作用和花粉管极性生长的独特细胞学过程,几十年来一直被作为研究这一系列精细细胞学过程的模式,但是其中关键事件的分子机制并不十分清楚。我们利用蛋白质组学技术鉴定了水稻成熟花粉三个不同组分(花粉外被蛋白、花粉外被/细胞壁相关和可释放蛋白、花粉内在蛋白)中表达的共322种蛋白质。其中,参与信号转导(10%)、细胞壁重塑和代谢(11%)、蛋白质代谢(14%)以及糖类和能量代谢(25%)的蛋白质被高度代表。并且很多是首次被在成熟花粉中得到鉴定的具有重要功能的蛋白质,包括蛋白激酶、受体激酶相互作用蛋白、GDP解聚抑制因子、含有C2结构域蛋白质和亲环蛋白等参与信号转导的蛋白质,eIF4A、线粒体加工肽酶、UFD1和AAA+ ATP酶等参与蛋白质合成、装配和降解的蛋白质,以及逆糖基化多肽、似纤维素合酶OsCsLF7等参与细胞壁重塑和代谢的蛋白质等。生物信息学分析表明,在我们鉴定的蛋白质中有11%的蛋白质功能未知并且不含有任何已知的功能结构域。另外,我们利用从头测序技术鉴定了5种新蛋白质。这些首次在花粉中被鉴定的蛋白质、未知功能蛋白质和新蛋白质在花粉萌发过程中的生物学功能值得进一步研究。进而,我们利用比较蛋白质组学技术获得了水稻花粉体外萌发过程中差异表达的160个鉴定结果(代表120种蛋白质),生物信息学分析表明它们隶属于13个功能类群。其中,参与细胞壁代谢(占24%,如UDP-葡萄糖焦磷酸化酶和第三类过氧化物酶)、蛋白质代谢(占11%,如eIF4A和20S蛋白酶体的亚基)、细胞骨架动力学(占8%,如肌动蛋白、微管蛋白和profilin)和胁迫反应(占7%,如抗坏血酸过氧化物酶)以及糖类和能量代谢(占24%)的蛋白质被高度代表。共有94个鉴定结果(73种蛋白质)在花粉萌发过程中表达丰度上调,包括几乎全部参与蛋白质代谢的蛋白质,多数参与细胞骨架动态变化和离子转运的蛋白质,以及部分参与糖类和能量代谢的蛋白质等。并且有53个鉴定结果(41种蛋白质)可能在萌发过程中被释放到培养基中更多还原

【Abstract】 As a highly reduced two-/three-cell organism, the pollen performs specialized functions to generate and carry sperms into the ovule by its polar elongation. Pollen has been the well model to investigate the sexual reproduction and cell polar growth in the past years. Yet molecular genetic basis of these functions is devoid now.Our proteomic analysis of mature pollen grains from Oryza sativa L. ssp japonica by a combination of 2DE with MALDI-TOF MS and ESI-Q-TOF MS/MS revealed that total 322 unique proteins expressed in three distinct protein fractions(pollen coat proteins, pollen coat/wall related and releasing proteins and pollen interior proteins). These proteins involved in signal transduction (10%), wall remodeling and metabolism (11%) and protein metabolism (14%), along with carbohydrate and energy metabolism (25%), were over-represented in pollen protein complement, some of which were important proteins identified in pollen firstly, mainly involving in signal transduction (such as protein kinases, receptor kinase-interacting proteins, GDP dissociation inhibitors, C2 domain-containing proteins, cyclophilins), protein synthesis, assembly and degradation (such as prohibitin, mitochondrial processing peptidase, putative UFD1, AAA+ ATPaes), and wall remodeling and metabolism (such as reversibly glycosylated polypeptides, cellulose synthase-like OsCsLF7). And bioinformatic analysis revealed that about 11% of the identified proteins were functionally unknown and did not contain any conserved domain associated with known activities. Also, five novel proteins were identified by de novo sequencing. The biological functions of pollen-expressed proteins including functionally unknown proteins and novel proteins need to be investigated further.Furthermore, a proteomic comparison between germinated pollen grains and mature pollen grains from rice resulted in the identification of 160 differentially-expressed identities (represented 120 unique proteins) upon germination, which could be assigned into 13 function categories by bioinformatic analyses. Of them, over-reperasentative proteins were those involved in the wall remodeling and metabolism (24% of the identities, such as UDP-glucose pyrophosphorylase and calss III peroxidase), protein metabolism (11%, such as eIF4A, members of 20S proteasome subunit), cytoskeleton dynamics (8%,更多还原