水牛孤雌激活胚胎蛋白质组学研究和功能验证

卵母细胞是合子基因组重编程的载体，其中的母源性物质是启动早期胚胎发育的关键物。本项目开展水牛MII期卵母细胞和孤雌胚胎的蛋白质组分析，定量分析差异蛋白质，寻找孤雌胚胎磷酸化修饰位点，筛选和验证重要母源性蛋白质的功能。包括：1）利用二维液相-质谱技术建立孤雌胚胎和MII期卵母细胞的蛋白质表达谱，为发现母源因子提供基础数据；2）利用外标定量技术，比较母源性蛋白质表达最强的孤雌卵裂胚与成熟卵母细胞的蛋白质表达差异，得到一批母源因子候选物信息；3）IMAC富集磷酸化肽，质谱分析和鉴定磷酸化位点，生物信息学分析以上表达量/修饰差异的蛋白质；4）RT-PCR、Western blot验证重要母源蛋白质在转录水平和翻译水平表达，细胞荧光免疫法分析蛋白质定位信息，初步解释其生物学功能。以上研究致力于发现早期胚胎中母源蛋白质的表达变化并验证功能，有助于深入认识哺乳动物早期胚胎发育的生物学调控途径。

Oocyte act as the genetic vector for zygote reprogramming, the maternal substance play a key role in preimplantation embryo development. In this study, Parthenogenetic activation embryo and MII oocyte of buffalo will be analyzed by proteomics methods and quantitative analysis of differential expressed proteins, The phosphorylation modification sites of parthenoactivation embryo will screened also, the important maternal proteins will be identifed in functional verification. This project involve four issues as follow, 1) LC-MS and Multi-dimensional Protein Identification Technology(MudPIT) was employed to construct the proteome profiling of Parthenoactivation cleavage (PC) and Metaphase II (MII) oocyte, and make the basis for maternal proteins discovery. 2) comparing the two stages (PC stage and MII stage when the maternal express highly) using TMT labelling technology, screening the maternal proteins (factors) candidates with important biology function. 3) enrich the phosphorylation peptide with IMAC method and analyis phosphorylation sites with LTQ-Orbitrap Mass spectrometer. 4) Verify the important candidate proteins with Realtime-PCR and Western blot and analyze the cell location with fluoroimmunoassay，preliminary interpretate the biological function of differential proteins. In short, by the way of a large scale of proteomics research and function verification, exploring the regulate rule of maternal protein in preimplantation embryo development, which has important significance of understanding biological pathway of mammalian preimplantation embryo development.