不同产羔性状绵羊卵巢组织差异表达蛋白质的筛选与分析

发布时间：2018-06-15 07:40

本文选题：绵羊 + 产羔性状　；参考：《甘肃农业大学》2015年博士论文

【摘要】：产羔性状是绵羊重要的经济性状之一,直接影响到养羊业生产的效率和成本,对养羊业的发展起到了关键性的作用。绵羊的产羔性状分为单羔性状、双羔性状和多羔性状三种,不同品种绵羊的产羔率差异较大。选育绵羊理想的产羔性状最有效的方法是掌握绵羊产羔性状的分子遗传机制和调控机理,从DNA分子角度寻找到产羔性状的主效基因和分子标记,进行分子选育。绵羊的产羔数是受排卵数控制的,在卵泡发育和排卵过程中,存在一系列的细胞调控机制,涉及到多种类型蛋白质相互作用。因此,要对绵羊产羔性状的调控机理有全面和深入的认识,就必须从整体、动态、调控途径上对作用于产羔性状的蛋白质进行研究,确定绵羊不同产羔性状的主效信号分子及其传递通路,揭示绵羊不同产羔性状的分子遗传机制和调控机理,为绵羊产羔性状分子标记选育和分子育种提供理论依据。本研究开展了用TCA/丙酮沉淀法和直接裂解法提取绵羊卵巢组织全蛋白方法的比较、筛选与优化研究,利用双向凝胶电泳和质谱技术,对乏情期(蒙古羊乏情期,无角陶赛特羊和小尾寒羊间情期,下同)、发情周期第1天和发情周期第11天的蒙古羊繁殖母羊(包括产单羔和双羔的母羊,简称单羔蒙古羊、双羔蒙古羊)、无角陶赛特羊繁殖母羊(包括产单羔和双羔的母羊,简称单羔无角陶赛特羊、双羔无角陶赛特羊)和产多羔小尾寒羊(简称多羔小尾寒羊)繁殖母羊的卵巢蛋白质组成及含量的动态变化进行分析,筛选不同产羔性状绵羊卵巢组织中差异表达的蛋白质,利用生物信息学方法对差异蛋白质进行亚细胞定位、GO分析(细胞组件、分子功能和生物过程)以及KEGG通路分析,构建差异蛋白质相互作用网络,以期探索绵羊不同产羔性状的分子作用机理及调控机制。⑴在相同电泳过程和操作步骤情况下获得的绵羊卵巢全蛋白2-DE图谱分析发现,直接裂解法对绵羊卵巢全蛋白的提取效果优于TCA/丙酮沉淀法,其中裂解液配方为7M尿素,2M硫脲,4%CHAPS,0.5%两性电解质pH 3-10,100mmol/L DTT,60 mmol/L Tris,20μL/mL蛋白酶抑制剂混合物时适合绵羊卵巢全蛋白质的提取。⑵运用双向凝胶电泳技术、生物质谱技术和蛋白质生物信息学对单羔蒙古羊、双羔蒙古羊、单羔无角陶赛特羊、双羔无角陶赛特羊和多羔小尾寒羊在乏情期、发情周期第1天和发情周期第11天卵巢组织差异蛋白进行筛选和分析,结果表明,共筛选到19种差异蛋白质,其中乏情期15种,发情周期第一天10种,发情周期第11天15种,差异蛋白质亚细胞定位预测结果显示19种差异蛋白质2种蛋白质位于分泌通路中(GDF9和glutathione S-transferase Mu 1-like isoform3),3种蛋白质位于线粒体中(myosin regulatory light chain MRCL3、glyceraldehyde-3-phosphate dehydrogenase和nitric oxide-inducible gene protein),其他14种蛋白质定位于其他部位;GO分析表明,基于GO分析中的细胞组件结果得到21个注释,在细胞质(cytoplasm)、细胞质基质(cytosol)、细胞核(nucleus)和钙离子复合物(calcium complex)呈富集状态,基于GO分析中的分子功能得到31个注释,在在ATP结合(ATP binding)、信号转导(signal transducer activity)、钙离子结合(calcium ion binding)、氧的转运活性(oxygen transporter activity)、生长因子活性(growth factor activity)、DNA结合(DNA binding)和碳水化合物结合(carbohydrate binding)呈现富集状态,基于GO分析中的生物过程得到35个注释,在氧化还原过程(oxidation-reduction process)、卵泡生长发育(ovarian follicle development)、信号转导(signal transduction)、BMP信号通路(BMP signaling pathway)、生长因子受体信号通路(growth factor receptor signaling pathway)、C21甾体激素的代谢过程(C21-steroid hormone metabolic process)和代谢过程(metabolic process)呈现富集状态;KEGG分析共得到23个注释,其中在氧化磷酸化通路(Oxidative phosphorylation pathway)、卵母细胞减数分裂(Oocyte meiosis)、TGF-β信号通路(TGF-beta signaling pathway)和GnRH信号通路(GnRH signaling pathway)呈现富集状态。⑶筛选与绵羊产羔性状相关的关键蛋白有:Calmodulin,GDF9,BMPR-1B,MTHFR和SOD-[Cu-Zn]-like。
[Abstract]:Lambing character is one of the important economic traits of sheep, which directly affects the efficiency and cost of sheep production, and plays a key role in the development of sheep industry. The lambing traits of sheep are divided into three traits, two lambs and multiple lambs, and the lambing rate of different sheep is different. The ideal lambing character of sheep is selected. The most effective method is to master the molecular genetic mechanism and regulation mechanism of lambing traits in sheep. From the angle of DNA, the main genes and molecular markers of lambing traits are found, and molecular breeding is carried out. The number of lambs in sheep is controlled by the number of ovulation. In the process of follicle development and ovulation, there are a series of cell regulation mechanisms involved in the process of ovarian follicle development and ovulation. Therefore, in order to have a comprehensive and deep understanding of the regulation mechanism of lambing traits in sheep, it is necessary to study the protein of lambing traits on the whole, dynamic and regulatory ways, to determine the main signal subdivisions and transmission pathways of different lambing traits in sheep, and to reveal the different lambing traits of sheep. Molecular genetic mechanism and regulatory mechanism provide a theoretical basis for molecular breeding and molecular breeding of lambing traits in sheep. This study carried out a comparison, screening and optimization of TCA/ acetone precipitation and direct lysis in sheep ovarian tissue, using two dimensional gel electrophoresis and mass spectrometry, for the Anoestrus (Mongolia sheep). The Mongolia sheep breeding ewes (including single lambs and double lambs, abbreviated as single lambs, Mongolia sheep, double lambs, Mongolia sheep), and non horns Dorset sheep breeding ewes (including single lambs and double Lambs), were found in the absence of estrus, with first days of estrus and eleventh days of estrous cycle. The dynamic changes in the protein composition and content of the ovaries of the two lambs and Small Tailed Han sheep were analyzed. The proteins expressed differently in the ovaries of the sheep were screened with different lambing traits. The subcellular localization of the differential proteins was carried out by bioinformatics method, and the GO analysis (fine) Cell components, molecular functions and biological processes) and KEGG pathway analysis to construct differential protein interaction networks in order to explore the molecular mechanism and regulation mechanism of different lambing traits in sheep. (1) analysis of the total protein 2-DE Atlas of ovine ovaries obtained under the same electrophoresis process and operation steps The extraction effect of total ovarian protein is better than that of TCA/ acetone precipitation, in which lysate formula is 7M urea, 2M thiourea, 4%CHAPS, 0.5% amphoteric electrolyte pH 3-10100mmol/L DTT, 60 mmol/L Tris, and 20 mu L/mL protease inhibitor mixture is suitable for the extraction of all protein in ovaries. 2. Bi-gel electrophoresis, biological mass spectrometry and eggs White matter bioinformatics was screened and analyzed for single lamb Mongolia sheep, double lamb Mongolia sheep, single lamb without horseback dovette sheep, double lambs without horseback Dorset sheep and multi lambs Small Tail Han sheep in the hypoestrus, first days of estrous cycle and eleventh days of estrous cycle, the results showed that 19 kinds of differential proteins were screened, among them, 15 species of hypoestrus were found. The first day of the estrous cycle is 10 species, and the estrous cycle is eleventh days 15. The difference protein subcellular location prediction results show that 19 kinds of protein 2 proteins are located in the secretory pathway (GDF9 and glutathione S-transferase Mu 1-like isoform3), and the 3 proteins are in the mitochondria (myosin regulatory light chain MRCL3, glyceraldehyde-3-phosphat). E dehydrogenase and nitric oxide-inducible gene protein), the other 14 proteins are located at other sites; GO analysis shows that the cell component results in the GO analysis are 21 annotations, in the cytoplasm (cytoplasm), the cytoplasmic matrix (cytosol), the nucleus (nucleus) and the calcium ion complex (calcium), based on the enrichment state. The molecular functions in the analysis are 31 annotations, in ATP binding (ATP binding), signal transduction (signal transducer activity), calcium ion binding (calcium ion binding), oxygen transport activity (oxygen transporter activity), growth factor activity and carbohydrate binding Binding) presents a state of enrichment, and 35 annotations are obtained in the biological process based on GO analysis, in the redox process (oxidation-reduction process), follicle growth and development (ovarian follicle development), signal transduction (signal transduction), BMP signaling pathway (BMP signaling), and growth factor receptor signaling pathway Eptor signaling pathway), the metabolic process (C21-steroid hormone metabolic process) and metabolic process (metabolic process) of C21 steroid were enriched, and 23 notes were obtained in the KEGG analysis, in which the oxidative phosphorylation pathway (Oxidative), oocyte meiosis, and beta signal TGF-beta signaling pathway and GnRH signaling pathway (GnRH signaling pathway) present enrichment status. (3) key proteins related to lamb traits of sheep are selected: Calmodulin, GDF9, BMPR-1B, MTHFR and SOD-[Cu-Zn]-like..
【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S826

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