水牛生殖细胞蛋白质组表达谱构建和定量差异分析

发布时间：2018-03-03 18:12

  本文选题：水牛　切入点：生殖细胞　出处：《广西大学》2016年博士论文　论文类型：学位论文

【摘要】：水牛(Bubalus bubalis)是我国宝贵的家畜品种,具有耐粗饲、饲料转化率高、乳品的营养价值高等特点,具有巨大的经济开发价值。但水牛繁殖效率低,生殖细胞成熟质量是影响水牛繁殖率的主要原因之一。生殖细胞成熟发育是复杂的生物学过程,这种复杂性主要表现在转录、翻译和翻译后修饰及调控网络上。蛋白质是生命功能的执行者,采用高通量的蛋白质组学技术可大规模发现蛋白质的表达和翻译后修饰,有助于从整体上揭示生殖细胞发育的蛋白质调控网络,筛选和鉴定关键基因或生物标志物。目前,生殖细胞的组学研究在模式生物中的开展较多,但在水牛上的报道相对较少,本文开展水牛生殖细胞及其发育内环境的蛋白质组研究,从蛋白质表达和修饰方面探究影响精子活力的因素,利用多组学关联分析获得卵母细胞成熟过程中的蛋白质表达变化和转录变化,为水牛的生殖生理研究提供新的研究点,为在分子水平上揭示精子和卵母细胞的发育和成熟打下理论基础。本文的主要研究结果如下:一、水牛生殖细胞(精子、卵母细胞)及微环境(精浆、卵泡液)蛋白质组表达谱构建和生物学信息学分析。利用MudPIT(Multi-dimensional Protein Identification Technology)蛋白质组学策略在精子和精浆中分别鉴定到2147种和864种蛋白质,在卵母细胞和卵泡液中分别鉴定到1710种和363种蛋白质。构建了包括蛋白质的登录号、名称、分子量、等电点、谱图等信息的水牛生殖细胞公共数据库。开源的公共数据库将为发育生物学研究提供有价值的资源。其次,对蛋白质表达谱进行生物信息学分析和数据挖掘,通过Gene Ontology分析,对精子、卵母细胞、精浆和卵泡液蛋白质的细胞定位、参与生物学过程和分子功能进行富集分析,筛选了一批与受精、减数分裂等有关的重要蛋白质候选物。通过KEGG数据库富集生殖细胞及其微环境涉及的各类生物学代谢通路,精子和卵母细胞中较多蛋白质参与各类能量代谢、合成代谢通路;精浆和卵泡液通过补体级联通路和蛋白酶体通路起到稳定内环境的作用。根据蛋白质的功能和作用关系,绘制了水牛精子、卵母细胞的蛋白质相互作用网络,发现了一批与配子发育有关的蛋白质互作事件。该部分研究丰富了水牛蛋白质组和功能基因信息,为阐明精子和卵母细胞的成熟机制提供了研究基础。二、精子活力是影响受精能力的关键因素之一,为了探寻精子活力的维持机制,比较了高、低活力精子的蛋白质表达和磷酸化修饰变化并进行实验验证。采用Percoll密度梯度离心分离高、低活力精子,分别提取总蛋白质,应用TMT(Tandem Mass Tag)外标定量法结合高分辨率质谱分析对水牛高、低活力精子的差异蛋白质开展了研究。结果表明,在低活力精子中筛选到145种表达显著差异的蛋白质,其中上调蛋白52种,下调蛋白93种,差异蛋白质变化总体呈现下降趋势。上调蛋白质主要涉及形态发生和调节细胞分化,下调蛋白质与转运、氧化还原反应、微管构成、精子动力、调节cAMP代谢过程、DNA甲基化调节等生物进程有关。通过磷酸化分析,在高活力精子中获得197种磷酸化蛋白质、384种磷酸化肽段和441个磷酸化位点,在低活力精子中获得178种磷酸化蛋白质、361种磷酸化肽段和424个磷酸化位点。磷酸化蛋白质在精子中普遍存在,涉及多个生物学事件和信号通路,暗示磷酸化修饰在精子功能的维持上起重要作用。比较高、低活力精子的磷酸化肽段,发现了 22个差异修饰位点异常,定位到的9种磷酸化蛋白质,包括激酶锚定蛋白3(AKAP3)、激酶锚定蛋白4(AKAP4)、外膜致密纤维蛋白1(ODF1)、纤维鞘互作蛋白(FSIP2)等结构性蛋白质。这些差异蛋白质的表达量变化或修饰变化共同说明低活力精子表现出能量代谢低效率、精子核鱼精蛋白不足、动力蛋白的下调和尾部结构蛋白磷酸化修饰变异。采用Western blot和RT-PCR验证了 6种差异蛋白质:鱼精蛋白1(PRAM1)、多配体糖蛋白(SDC2)、细胞支架蛋白3(TEKT3)、激酶锚定蛋白3(AKAP3)、螺旋卷曲蛋白40(CCDC40)、异柠檬酸脱氢酶(IDH1),它们的表达量变化均与质谱定量结果一致,RT-PCR证实SDC2、TEKT3和IDH1蛋白质的mRNA与蛋白质的表达相关性较低,说明这些蛋白质变化受到翻译调控影响。三、卵母细胞在体外成熟的分子机制尚不完全清楚,为获得水牛卵母细胞成熟过程的核质变化,比较了卵母细胞成熟培养前后的蛋白质表达差异,结合转录组数据进行系统生物学分析。首先,应用TMT外标定量结合质谱技术比较了水牛成熟培养前后卵母细胞的蛋白质变化,以成熟前卵母细胞作为对照组,成熟后的卵母细胞中鉴定到62种差异蛋白质,38种蛋白质表达上调,24种表达下调,差异蛋白质涉及能量合成、RNA剪切、转运等生物过程。Western blot验证了 HSP60和GEMIN8蛋白质表达变化。为发现相关基因在转录水平的表达量变化,通过高通量测序获得成熟前、成熟后卵母细胞转录本,与参考基因组比对率分别为86.4%和86.8%,与转录本比对率分别为22.4%和23.7%。比较两个转录组文库的FPKM值,发现了2667个差异表达基因,1695个基因在成熟卵母细胞中表达量下调,972个基因上调,转录组差异基因数量明显高于蛋白质水平。COG分析表明,转录、复制重组、信号传导机制、核糖体结构、翻译后修饰等是差异基因涉及的主要事件。差异基因与多种信号通路有关联,与差异蛋白质的功能聚类分析有较大不同。将转录组序列与蛋白质组序列进行BLASTx比对,将共同表达的unigene进行关联分析,相关系数r = 0.49,说明卵母细胞在成熟过程中转录和翻译的相关性较低。在筛选到的62个差异表达蛋白质中,27个差异蛋白质可在转录组中得到关联,相关系数r = 0.57,其中14种蛋白质的基因转录和蛋白质表达均呈现显著性变化。综上所述,本试验将高通量的蛋白质组学和差异磷酸化分析的技术体系应用于水牛精子活力研究,有助于阐明蛋白质变化与精子活力维持的关系。将多组学技术用于卵母细胞发育研究,发现卵母细胞成熟过程的基因和蛋白质表达变化,对于探讨卵母细胞成熟机制和提高IVF效率具有重要价值。
[Abstract]:Buffalo (Bubalus bubalis) is our precious animal breed, with resistant coarse feed, feed conversion rate, high nutritional value of dairy products, has great value in economic development. But buffalo low reproductive efficiency, germ cell maturation is one of the main reasons affecting the quality of buffalo. The reproductive rate of germ cell maturation is a biological process the complex, this complexity is mainly manifested in the modification and regulation of transcription, translation and post translation network. The protein is a vital function executor, using high-throughput proteomic technology can be found in large scale expression and translation of protein modifications, contribute to the protein regulatory network revealed germ cell development as a whole, and screening identification of key genes or biomarkers. At present, the study on germ cells in model organisms are carried out, but in Buffalo's reports are relatively small, this paper carried out Proteomic study of germ cell development and buffalo internal environment, from the protein expression and modification factors to explore the impact of sperm motility, using multi omics correlation analysis to obtain the process of oocyte maturation protein expression and transcriptional changes in physiological studies provide new research points for buffalo's sperm and oocytes for reproduction. The development and maturation of cells to lay a theoretical foundation to reveal at the molecular level. The main results are as follows: first, buffalo germ cells (sperm and oocytes) and micro environment (seminal plasma and follicular fluid) proteome expression profile construction and bioinformatics analysis. Using MudPIT (Multi-dimensional Protein Identification Technology) protein group learning strategies in the sperm and seminal plasma were identified to 2147 species and 864 kinds of proteins in oocyte and follicular fluid were identified to 1710 species and 363 species of protein structure. Built including protein accession number, name, molecular weight and isoelectric point, buffalo germ cell public database spectrum information. The public database open source will provide valuable resources for the development of biological research. Secondly, the spectrum of bioinformatics analysis and data mining of protein expression by Gene Ontology analysis on. Sperm, oocyte, cellular localization of seminal plasma and follicular fluid protein, involved in the biological process and molecular function enrichment analysis, screening a number of important candidate proteins and fertilization, meiosis related. Through KEGG database all kinds of biological metabolic pathway enrichment of germ cells and their microenvironment involved in energy metabolism, all kinds of more proteins involved in sperm and oocytes, synthetic metabolic pathways; seminal plasma and follicular fluid by complement cascade and proteasome pathway plays a stable internal environment. According to the function and role of protein, rendering buffalo sperm, oocyte protein interaction network, found a number of gametophyte development related protein interaction events. The study enriches the buffalo proteome and functional gene information, and provides the basis for clarifying the mechanism of mature sperm and oocytes. Two, sperm motility is one of the key factors affecting the fertilization ability, in order to maintain the possible mechanism of sperm motility, compared high, low sperm protein expression and phosphorylation changes and verified by the experiment. Using Percoll density gradient centrifugation, low motility, total proteins were extracted from TMT (Tandem Mass, application Tag) quantitative method combined with high-resolution mass spectrometry analysis of Buffalo high, low sperm proteins were studied. The results showed that the screened 145 in low activity in sperm A significant difference in the expression of proteins, including 52 up-regulated proteins and 93 down regulated proteins, proteins change the overall downward trend. Up regulation of protein mainly involves the morphogenesis and cell differentiation, and down-regulation of protein transport, redox reaction, microtubules, sperm motility, regulating cAMP metabolism, DNA methylation regulation and other biological processes. Through phosphorylation analysis, obtained 197 kinds of phosphorylated proteins in high motility, 384 phosphopeptides and 441 phosphorylation sites, 178 protein phosphorylation in low motility, 361 phosphopeptides and 424 phosphorylation sites. The phosphorylated proteins exist in sperm, involving multiple biological events and signaling pathways, suggesting that phosphorylation is maintained in sperm function play an important role. The higher the phosphopeptide low motility, found 22 differences 9 kinds of abnormal modification sites, phosphate to protein localization, including kinase anchoring protein 3 (AKAP3), a kinase anchoring protein 4 (AKAP4), outer dense fiber protein 1 (ODF1), fiber sheath interacting protein (FSIP2) and other structural proteins. The expression changes or modifications of these proteins together low sperm motility showed a low efficiency of energy metabolism, sperm protamine deficiency, and down-regulation of dynein tail structure protein phosphorylation by Western and blot mutation. RT-PCR tested 6 proteins: protamine 1 (PRAM1), multiple ligand glycoprotein (SDC2), 3 (TEKT3) cell scaffold protein kinase. Anchor protein 3 (AKAP3), 40 (CCDC40) protein coiled, isocitrate dehydrogenase (IDH1), expression of them are consistent with mass spectrometry quantitative results, confirmed by RT-PCR SDC2, TEKT3 and IDH1 protein and mRNA protein expression correlated These changes are low, indicating the protein translation regulation. Three oocytes in vitro maturation in molecular mechanism is not completely understood, as the change process of maturation of buffalo karyoplasms oocytes, the oocytes maturation and protein expression differences, combined transcriptome analysis of systems biology. First of all, the application of TMT quantitative mass spectrometry combined with comparison of protein changes before and after cultured oocytes buffalo mature, in the mature oocyte as the control group, after the mature oocytes identified 62 different proteins, 38 proteins up-regulated and 24 down regulated the expression of proteins involved in energy synthesis, RNA shear, transport and other biological processes.Western blot validated HSP60 and GEMIN8 protein expression was found. Expression of related genes at the transcriptional level, obtained by high-throughput sequencing Mature, mature oocyte transcripts, and reference genome alignment rates were 86.4% and 86.8%, and the ratio of transcription rates were 22.4% and two 23.7%. compared the transcriptome library FPKM, found 2667 differentially expressed genes, 1695 gene expression in oocytes matured in 972. The number of up-regulated genes, gene transcription was significantly higher than that of group differences in protein level.COG analysis showed that the recombinant replication, transcription, signal transduction mechanism, ribosome structure, post-translational events are the main differences in genes involved. Different genes are associated with several signal pathways, and clustering analysis of protein function differences are quite different. The transcription sequence and protein sequence alignment with BLASTx, CO expressed UniGene for correlation analysis, correlation coefficient r = 0.49, indicating a correlation between oocyte transcription and translation during the ripening process Low. In 62 different screening of the protein expression in 27 different proteins can be associated in the transcriptome, the correlation coefficient r = 0.57, the expression of mRNA and protein of 14 proteins showed significant changes. In conclusion, this experiment will study the proteome high-throughput analysis and differential phosphorylation the technical system used in buffalo sperm motility, helps to elucidate the relationship between protein changes and sperm motility maintained. Multi omics technologies used to study the development of oocytes, discovery of genes and proteins during oocyte maturation and the expression changes, to explore the mechanism of oocyte maturation and improve the efficiency of IVF is of great value.

【学位授予单位】：广西大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823.83
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本文编号：1562131