不同发育期绵羊睾丸差异蛋白质组学研究

发布时间：2018-03-01 18:55

本文关键词： 绵羊睾丸蛋白质组学双向电泳(2-DE)　出处：《甘肃农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：雄性生殖能力对动物繁殖和养殖业至关重要。睾丸作为雄性动物重要的生殖器官,具有产生精子和分泌雄激素的功能。这一复杂过程的完成需要多种蛋白质相对精确协调地表达和调控。因此,应用蛋白质组学方法来探索睾丸中蛋白质的组成具有重要意义。本研究以绵羊睾丸为实验材料,首先建立和优化绵羊睾丸蛋白质组学双向电泳体系;之后应用蛋白质组学、质谱和生物信息学技术比较分析了不同发育阶段绵羊睾丸蛋白质组成和表达的差异情况;最后应用q RT-PCR、Western blot和免疫组化方法对不同发育期绵羊睾丸中部分差异蛋白质表达情况进行验证。主要研究结果如下:1.绵羊睾丸蛋白质双向电泳体系的建立及优化应用17 cm p H 3—10的非线性IPG胶条进行双向电泳,比较了蛋白不同提取方法、上样量、聚焦时间和分离胶浓度对绵羊睾丸蛋白质双向电泳的影响。结果表明,三氯乙酸(TCA)/丙酮沉淀法提取绵羊睾丸组织总蛋白获得的蛋白点最多,上样量750μg,聚焦时间75000 Vh或更高,12%分离胶对分离后的2-DE图谱较好。采用优化后的条件,可以得到蛋白点数多、背景清晰的2-DE图谱。2.不同发育期绵羊睾丸蛋白质组学研究应用2-DE技术对绵羊5个不同发育期(0、2、5、12和24月龄)睾丸蛋白质进行检测,分别检测到382、522、567、596和671个蛋白点。应用PDquest 8.0图像分析软件共筛选出45个差异蛋白点,经过MALDI-TOF-TOF质谱鉴定,最终成功鉴定出37种蛋白质。使用Blast2GO对每一种蛋白质的生物学过程、细胞组成和分子功能进行详细注释,这些蛋白质主要参与了细胞过程和单有机体过程‖、细胞和细胞器组成‖及结合和催化活性‖。3.绵羊睾丸差异蛋白质的验证应用q RT-PCR方法对15个蛋白质在绵羊睾丸5个不同发育期的转录水平进行检测,其中6个基因与蛋白水平的表达趋势一致,9个基因与蛋白水平的表达趋势不一致。应用Western blot和免疫组化方法对4个蛋白质进行检测,其表达趋势与2-DE结果一致。
[Abstract]:Male reproductive ability is crucial to animal reproduction and breeding. Testicles are important reproductive organs for male animals. The ability to produce sperm and secrete androgen. The completion of this complex process requires the relatively precise and coordinated expression and regulation of multiple proteins. It is important to explore the protein composition in testis by using proteomics method. In this study, a two-dimensional electrophoresis system of testis proteomics was established and optimized using sheep testis as experimental materials, and then proteomics was used. Mass spectrometry and bioinformatics were used to compare and analyze the differences of protein composition and expression in sheep testis at different stages of development. Finally, the expression of some differentially expressed proteins in the testis of sheep at different stages of development was verified by Q RT-PCR blot and immunohistochemistry. The main results are as follows: 1. The establishment and optimization of two-dimensional electrophoresis system of testis protein in sheep. Two-dimensional electrophoresis was carried out with 17cm pH3-10 nonlinear IPG strips. The effects of different extraction methods, sample amount, focusing time and gel concentration on the two-dimensional electrophoresis of sheep testis protein were compared. The results showed that the total protein extracted from sheep testis by TCA / acetone precipitation method had the most protein points. The concentration of sample was 750 渭 g, the focusing time was 75000 Vh or higher than 12%, and the 2-DE map was better after separation. Background 2-DE map .2.The 2-DE technique was used to detect the testis proteins of 5 sheep at different developmental stages (12 and 24 months old). 382,522,567,596 and 671 protein spots were detected respectively. A total of 45 differential protein spots were screened by PDquest 8.0 image analysis software, and 37 proteins were successfully identified by MALDI-TOF-TOF mass spectrometry. The biological process of each protein was determined by Blast2GO. Cell composition and molecular function are explained in detail, These proteins are mainly involved in the process of cell and single organism, the composition of cell and organelle, the binding and catalytic activity of these proteins. Validation of differential proteins in Sheep testis using Q RT-PCR method for 15 proteins. The transcriptional levels of the ovine testis at five different stages of development were measured. The expression trend of 6 genes and protein levels was the same, while that of 9 genes was not the same. Four proteins were detected by Western blot and immunohistochemistry. The results of 2-DE were consistent with the results of 2-DE.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S826

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