本文選題：水牛　切入點：生殖細胞　出處：《廣西大學》2016年博士論文　論文類型：學位論文

【摘要】：水牛(Bubalus bubalis)是我國寶貴的家畜品種,具有耐粗飼、飼料轉化率高、乳品的營養(yǎng)價值高等特點,具有巨大的經(jīng)濟開發(fā)價值。但水牛繁殖效率低,生殖細胞成熟質量是影響水牛繁殖率的主要原因之一。生殖細胞成熟發(fā)育是復雜的生物學過程,這種復雜性主要表現(xiàn)在轉錄、翻譯和翻譯后修飾及調控網(wǎng)絡上。蛋白質是生命功能的執(zhí)行者,采用高通量的蛋白質組學技術可大規(guī)模發(fā)現(xiàn)蛋白質的表達和翻譯后修飾,有助于從整體上揭示生殖細胞發(fā)育的蛋白質調控網(wǎng)絡,篩選和鑒定關鍵基因或生物標志物。目前,生殖細胞的組學研究在模式生物中的開展較多,但在水牛上的報道相對較少,本文開展水牛生殖細胞及其發(fā)育內環(huán)境的蛋白質組研究,從蛋白質表達和修飾方面探究影響精子活力的因素,利用多組學關聯(lián)分析獲得卵母細胞成熟過程中的蛋白質表達變化和轉錄變化,為水牛的生殖生理研究提供新的研究點,為在分子水平上揭示精子和卵母細胞的發(fā)育和成熟打下理論基礎。本文的主要研究結果如下:一、水牛生殖細胞(精子、卵母細胞)及微環(huán)境(精漿、卵泡液)蛋白質組表達譜構建和生物學信息學分析。利用MudPIT(Multi-dimensional Protein Identification Technology)蛋白質組學策略在精子和精漿中分別鑒定到2147種和864種蛋白質,在卵母細胞和卵泡液中分別鑒定到1710種和363種蛋白質。構建了包括蛋白質的登錄號、名稱、分子量、等電點、譜圖等信息的水牛生殖細胞公共數(shù)據(jù)庫。開源的公共數(shù)據(jù)庫將為發(fā)育生物學研究提供有價值的資源。其次,對蛋白質表達譜進行生物信息學分析和數(shù)據(jù)挖掘,通過Gene Ontology分析,對精子、卵母細胞、精漿和卵泡液蛋白質的細胞定位、參與生物學過程和分子功能進行富集分析,篩選了一批與受精、減數(shù)分裂等有關的重要蛋白質候選物。通過KEGG數(shù)據(jù)庫富集生殖細胞及其微環(huán)境涉及的各類生物學代謝通路,精子和卵母細胞中較多蛋白質參與各類能量代謝、合成代謝通路;精漿和卵泡液通過補體級聯(lián)通路和蛋白酶體通路起到穩(wěn)定內環(huán)境的作用。根據(jù)蛋白質的功能和作用關系,繪制了水牛精子、卵母細胞的蛋白質相互作用網(wǎng)絡,發(fā)現(xiàn)了一批與配子發(fā)育有關的蛋白質互作事件。該部分研究豐富了水牛蛋白質組和功能基因信息,為闡明精子和卵母細胞的成熟機制提供了研究基礎。二、精子活力是影響受精能力的關鍵因素之一,為了探尋精子活力的維持機制,比較了高、低活力精子的蛋白質表達和磷酸化修飾變化并進行實驗驗證。采用Percoll密度梯度離心分離高、低活力精子,分別提取總蛋白質,應用TMT(Tandem Mass Tag)外標定量法結合高分辨率質譜分析對水牛高、低活力精子的差異蛋白質開展了研究。結果表明,在低活力精子中篩選到145種表達顯著差異的蛋白質,其中上調蛋白52種,下調蛋白93種,差異蛋白質變化總體呈現(xiàn)下降趨勢。上調蛋白質主要涉及形態(tài)發(fā)生和調節(jié)細胞分化,下調蛋白質與轉運、氧化還原反應、微管構成、精子動力、調節(jié)cAMP代謝過程、DNA甲基化調節(jié)等生物進程有關。通過磷酸化分析,在高活力精子中獲得197種磷酸化蛋白質、384種磷酸化肽段和441個磷酸化位點,在低活力精子中獲得178種磷酸化蛋白質、361種磷酸化肽段和424個磷酸化位點。磷酸化蛋白質在精子中普遍存在,涉及多個生物學事件和信號通路,暗示磷酸化修飾在精子功能的維持上起重要作用。比較高、低活力精子的磷酸化肽段,發(fā)現(xiàn)了 22個差異修飾位點異常,定位到的9種磷酸化蛋白質,包括激酶錨定蛋白3(AKAP3)、激酶錨定蛋白4(AKAP4)、外膜致密纖維蛋白1(ODF1)、纖維鞘互作蛋白(FSIP2)等結構性蛋白質。這些差異蛋白質的表達量變化或修飾變化共同說明低活力精子表現(xiàn)出能量代謝低效率、精子核魚精蛋白不足、動力蛋白的下調和尾部結構蛋白磷酸化修飾變異。采用Western blot和RT-PCR驗證了 6種差異蛋白質:魚精蛋白1(PRAM1)、多配體糖蛋白(SDC2)、細胞支架蛋白3(TEKT3)、激酶錨定蛋白3(AKAP3)、螺旋卷曲蛋白40(CCDC40)、異檸檬酸脫氫酶(IDH1),它們的表達量變化均與質譜定量結果一致,RT-PCR證實SDC2、TEKT3和IDH1蛋白質的mRNA與蛋白質的表達相關性較低,說明這些蛋白質變化受到翻譯調控影響。三、卵母細胞在體外成熟的分子機制尚不完全清楚,為獲得水牛卵母細胞成熟過程的核質變化,比較了卵母細胞成熟培養(yǎng)前后的蛋白質表達差異,結合轉錄組數(shù)據(jù)進行系統(tǒng)生物學分析。首先,應用TMT外標定量結合質譜技術比較了水牛成熟培養(yǎng)前后卵母細胞的蛋白質變化,以成熟前卵母細胞作為對照組,成熟后的卵母細胞中鑒定到62種差異蛋白質,38種蛋白質表達上調,24種表達下調,差異蛋白質涉及能量合成、RNA剪切、轉運等生物過程。Western blot驗證了 HSP60和GEMIN8蛋白質表達變化。為發(fā)現(xiàn)相關基因在轉錄水平的表達量變化,通過高通量測序獲得成熟前、成熟后卵母細胞轉錄本,與參考基因組比對率分別為86.4%和86.8%,與轉錄本比對率分別為22.4%和23.7%。比較兩個轉錄組文庫的FPKM值,發(fā)現(xiàn)了2667個差異表達基因,1695個基因在成熟卵母細胞中表達量下調,972個基因上調,轉錄組差異基因數(shù)量明顯高于蛋白質水平。COG分析表明,轉錄、復制重組、信號傳導機制、核糖體結構、翻譯后修飾等是差異基因涉及的主要事件。差異基因與多種信號通路有關聯(lián),與差異蛋白質的功能聚類分析有較大不同。將轉錄組序列與蛋白質組序列進行BLASTx比對,將共同表達的unigene進行關聯(lián)分析,相關系數(shù)r = 0.49,說明卵母細胞在成熟過程中轉錄和翻譯的相關性較低。在篩選到的62個差異表達蛋白質中,27個差異蛋白質可在轉錄組中得到關聯(lián),相關系數(shù)r = 0.57,其中14種蛋白質的基因轉錄和蛋白質表達均呈現(xiàn)顯著性變化。綜上所述,本試驗將高通量的蛋白質組學和差異磷酸化分析的技術體系應用于水牛精子活力研究,有助于闡明蛋白質變化與精子活力維持的關系。將多組學技術用于卵母細胞發(fā)育研究,發(fā)現(xiàn)卵母細胞成熟過程的基因和蛋白質表達變化,對于探討卵母細胞成熟機制和提高IVF效率具有重要價值。
[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.

【學位授予單位】：廣西大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S823.83
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本文編號：1562131