【作者】 张玲；

【导师】 谢锦云； 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 第一部分金鱼雌核发育单倍体胚胎无腔眼和正常眼胚胎的蛋白质组研究在金鱼雌核发育单倍体胚胎发育过程中，一些对发育起关键作用的基因未能被选择表达或表达受阻而导致单倍体发育异常。在单倍体胚胎发育过程中出现了正常眼，泡状眼，无腔眼三种表型，经统计分析表明，单倍体发育的胚胎数目以正常眼：泡状眼：无腔眼为1：2：1的比例分配。我们已研究证明Vsx1基因在单倍体胚胎中表达受阻，而导致了单倍体胚胎发育异常，其中正常眼单倍体胚胎中Vsx1基因能够表达，无腔眼中Vsx1的基因不能表达，泡状眼中两个基因一个表达，一个未表达。与二倍体相比，Vsx1基因在单倍体胚胎中未能被选择表达，可能与单倍体胚胎发育异常有关。为了揭示金鱼雌核发育单倍体胚胎发育过程中的这种发育异常的机制，本文以遗传背景一致的金鱼雌核发育单倍体形成的正常眼和无腔眼发育阶段的胚胎为材料，通过蛋白质表达水平上进行差异分析，试图探讨影响金鱼眼睛发育的基因表达调控机理。首先提取胚胎的全蛋白质，用SDS-PAGE和二维聚丙烯酰胺凝胶电泳的方法，进行蛋白质点的分离，获得了分辨率较高的凝胶图谱。利用PDQUEST 2-D分析软件对扫描后的凝胶图象进行了处理，斑点检测，匹配和量化分析，得到差异蛋白质点，选取其中分辨好的差异蛋白质点进行原位胰蛋白酶酶解，再经MALDI-TOF-TOF和Q-TOF的MS／MS分析，通过MASCOT数据库及MS BLAST同源性搜索，一共鉴定到59个差异蛋白质点。通过生物信息学分析方法对这些差异蛋白质点的功能分析，发现了与眼睛发育，神经发育，细胞分化，体节形成，信号传导等相关的蛋白质。此外，通过Western blotting实验，某些重要差异蛋白质已得到了讲一步的验证。第二部分基于质谱的BLAST方法在未知基因组的金鱼蛋白质鉴定中的应用未知基因组及蛋白质序列数据库有限的物种的蛋白质组学分析是当前一些非模式生物物种蛋白质组学研究领域的瓶颈之一。基于同源性搜索的blast方法(MS BLAST)，是近年新发展起来的一种用于未知基因组的蛋白质鉴定的搜索工具，已成功应用于许多未知基因组物种的蛋白质鉴定。SPITC化学辅助方法是本实验室建立的一种改进的de novo质谱测序方法。我们采用MS BLAST方法对经Mascot软件数据库搜索未能鉴定到的19个金鱼胚胎蛋白质进行鉴定，其中12个蛋白质是直接测序后进行MS BLAST搜索得到的结果，另外7个蛋白质是联合MS BLAST和SPITC衍生方法得到的鉴定结果。实验结果证明了采用Ms BLAST方法进行蛋白质的跨物种鉴定具有可行性和可靠性，给蛋白质的跨物种鉴定提供了一条新的途径。更多还原

【Abstract】 PartⅠ: Proteome comparative analysis of gynogenetic haploid embryos with normal eyes and cupless eyes of goldfishOur previous work indicated that obstruction of some protein expression cause the abnormal development of haploid embryos, such as the protein Vsx1(Huang LY et al). Further studies on Vsx1 confirmed the diploid development regulatory mechanism of gene expression during embryonic morphogenesis of goldfish (Luo et al) .The two genes of Vsx1 are expressed in gynogenetic haploid embryos with normal eyes of goldfish, while not with cupless eyes at all.To better understand the molecular events underlying the processes occurring at the embryos developing of gynogenetic haploid and elucidate the underlying molecular mechanism of abnormal development in haploid embryos, we developed a proteome comparative analysis of the gynogenetic haploid embryos with normal eyes and cupless eyes of goldfish in the same stage to disclose more differentially expressed proteins as Vsx1. The results indicated protein profiles of embryos with normal eyes and with cupless eyes exhibited some differences. Fifty-two protein spots were directly identified by mass spectrometry and mascot search. Seven protein spots were identified by sequence similarity search using mass spectrum driven BLAST (MS BLAST). Western blot analysis was further carried out to verify the different expression of four important proteins, which offered different significantly gene expression during embryos development between haploid embryos with normal eyes and with cupless eyes.PartⅡ: The application of Mass Spectrometry driven BLAST in protein identification of goldfish embryosProtein identification is a significant bottleneck in proteomic researches of some species with unknown genomes or uncompleted protein database at present. Mass Spectrometry driven BLAST (MS BLAST) is a sequence similarity searching tool, which has been developed in recent years and successfully applied to the identification of protein from species with unsequenced genomes. SPITC derivation is an improved de novo sequencing method by Mass Spectrometry. Combining these two methods, nineteen proteins of goldfish embryos not identified by Mascot searching were successfully identified by MS BLAST. Among the nineteen proteins, twelve were identified by MS BLAST searching, while the other seven by MS BLAST searching based on de novo sequencing of SPITC derivated peptides. This strategy has been proved feasible and reliable for protein identification of goldfish embryos, and provides a new pathway for protein cross-species identification of species with unsequenced genomes.更多还原