本文關鍵詞： 綿羊 睪丸 蛋白質組學 雙向電泳(2-DE)　出處：《甘肅農業(yè)大學》2016年碩士論文　論文類型：學位論文

【摘要】：雄性生殖能力對動物繁殖和養(yǎng)殖業(yè)至關重要。睪丸作為雄性動物重要的生殖器官,具有產生精子和分泌雄激素的功能。這一復雜過程的完成需要多種蛋白質相對精確協調地表達和調控。因此,應用蛋白質組學方法來探索睪丸中蛋白質的組成具有重要意義。本研究以綿羊睪丸為實驗材料,首先建立和優(yōu)化綿羊睪丸蛋白質組學雙向電泳體系;之后應用蛋白質組學、質譜和生物信息學技術比較分析了不同發(fā)育階段綿羊睪丸蛋白質組成和表達的差異情況;最后應用q RT-PCR、Western blot和免疫組化方法對不同發(fā)育期綿羊睪丸中部分差異蛋白質表達情況進行驗證。主要研究結果如下:1.綿羊睪丸蛋白質雙向電泳體系的建立及優(yōu)化應用17 cm p H 3—10的非線性IPG膠條進行雙向電泳,比較了蛋白不同提取方法、上樣量、聚焦時間和分離膠濃度對綿羊睪丸蛋白質雙向電泳的影響。結果表明,三氯乙酸(TCA)/丙酮沉淀法提取綿羊睪丸組織總蛋白獲得的蛋白點最多,上樣量750μg,聚焦時間75000 Vh或更高,12%分離膠對分離后的2-DE圖譜較好。采用優(yōu)化后的條件,可以得到蛋白點數多、背景清晰的2-DE圖譜。2.不同發(fā)育期綿羊睪丸蛋白質組學研究應用2-DE技術對綿羊5個不同發(fā)育期(0、2、5、12和24月齡)睪丸蛋白質進行檢測,分別檢測到382、522、567、596和671個蛋白點。應用PDquest 8.0圖像分析軟件共篩選出45個差異蛋白點,經過MALDI-TOF-TOF質譜鑒定,最終成功鑒定出37種蛋白質。使用Blast2GO對每一種蛋白質的生物學過程、細胞組成和分子功能進行詳細注釋,這些蛋白質主要參與了 細胞過程和單有機體過程‖、 細胞和細胞器組成‖及 結合和催化活性‖。3.綿羊睪丸差異蛋白質的驗證應用q RT-PCR方法對15個蛋白質在綿羊睪丸5個不同發(fā)育期的轉錄水平進行檢測,其中6個基因與蛋白水平的表達趨勢一致,9個基因與蛋白水平的表達趨勢不一致。應用Western blot和免疫組化方法對4個蛋白質進行檢測,其表達趨勢與2-DE結果一致。
[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.
【學位授予單位】：甘肅農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S826

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