發(fā)布時間：2018-05-02 04:11

  本文選題：牦牛 + 單細(xì)胞測序��； 參考：《西南民族大學(xué)》2017年碩士論文

【摘要】：牦牛是我國青藏高原特有的牛種,是當(dāng)?shù)啬撩裰饕纳钌a(chǎn)資料,但其繁殖和生產(chǎn)性能低下,阻礙著高原地區(qū)畜牧業(yè)的發(fā)展。卵母細(xì)胞的體外成熟培養(yǎng)是體外受精、胚胎移植等繁殖技術(shù)的基礎(chǔ)環(huán)節(jié)。與體內(nèi)成熟相比,體外成熟的卵母細(xì)胞進(jìn)行受精后所獲得胚胎的發(fā)育能力較低,胚胎移植后也面臨著妊娠率低、易流產(chǎn)、活產(chǎn)率低等問題。大量研究證明卵母細(xì)胞周圍的顆粒細(xì)胞在卵母細(xì)胞的成熟及發(fā)育中起著重要的角色,該類細(xì)胞可以通過旁分泌途徑及縫隙連接為卵母細(xì)胞直接提供營養(yǎng)、代謝小分子、第二信使等,間接影響卵母細(xì)胞的減數(shù)分裂、胞質(zhì)成熟、受精乃至后續(xù)胚胎的發(fā)育潛能。因此本研究以牦牛顆粒細(xì)胞為研究對象,挖掘了GⅤ期、MⅠ期、MⅡ期的轉(zhuǎn)錄組信息,分析其生物學(xué)信息,并對細(xì)胞周期蛋白D1和D2(CCND1和CCND2)基因與卵母細(xì)胞發(fā)育關(guān)聯(lián)性,結(jié)果如下:1、利用地衣紅染色方法從核形態(tài)上確定了卵母細(xì)胞成熟培養(yǎng)0 h、12 h、22 h時分別獲得了GⅤ期、MⅠ期、MⅡ期的卵母細(xì)胞。2、對三個時期卵母細(xì)胞周圍的顆粒細(xì)胞轉(zhuǎn)錄組測序結(jié)果分析顯示,GⅤ期、MⅠ期、MⅡ期分別有7540、8491、7520個轉(zhuǎn)錄本;其中GⅤ期與MⅠ期差異表達(dá)基因有318個上調(diào),304個下調(diào);MⅠ期與MⅡ期差異表達(dá)基因有141個上調(diào),334個下調(diào);GⅤ期與MⅡ期差異表達(dá)基因191個上調(diào),88個下調(diào);qPCR驗證結(jié)果與轉(zhuǎn)錄組數(shù)據(jù)結(jié)果一致,表明轉(zhuǎn)錄組測序數(shù)據(jù)可靠。3、對CCND1和CCND2進(jìn)行免疫組化定位、免疫熒光定性、實時熒光定量和免疫印跡分析結(jié)果顯示:CCND1和CCND2蛋白在顆粒細(xì)胞中的免疫組化均呈陽性,且GⅤ期顆粒細(xì)胞中的mRNA和蛋白的表達(dá)水平顯著高于MⅠ期和MⅡ期。4、與卵母細(xì)胞成熟和發(fā)育的關(guān)聯(lián)性研究結(jié)果顯示CCND1和CCND2與牦牛卵母細(xì)胞體外成熟率和受精后的囊胚率呈正相關(guān)。綜上,本研究通過單細(xì)胞轉(zhuǎn)錄組研究首次獲得了牦牛卵母細(xì)胞成熟過程中其顆粒細(xì)胞的基因表達(dá)譜。與牦牛卵母細(xì)胞成熟和發(fā)育能力的關(guān)聯(lián)性分析表明CCND1和CCND2與卵母細(xì)胞發(fā)育潛能成正相關(guān)。由此推測CCND1和CCND2可作為卵母細(xì)胞質(zhì)量和發(fā)育潛能評估的生物標(biāo)記,為篩選優(yōu)質(zhì)卵母細(xì)胞、改善體外成熟體系以及牦牛卵母細(xì)胞發(fā)育機(jī)制研究奠定基礎(chǔ)。
[Abstract]:Yak is a special breed of cattle in the Qinghai Tibet Plateau of China. It is the main production data of local herdsmen, but its reproduction and production performance is low, which hinders the development of animal husbandry in the plateau. In vitro maturation and culture of oocyte is the basic link of in vitro fertilization, embryo transfer and other reproductive techniques. The embryonic development ability of the cells after fertilization is low, and the embryo transfer is also faced with low pregnancy rate, easy abortion and low survival rate. A large number of studies have shown that the granulosa cells around oocytes play an important role in the maturation and development of oocytes, and this kind of cells can be connected to the oocytes through paracrine pathways and gaps. Cells directly provide nutrition, metabolic small molecules, second messenger and so on, which indirectly affect meiosis, maturation of the oocytes, fertilization and the developmental potential of subsequent embryos. Therefore, this study took yak granular cells as the research object, excavated the transcriptional information of G V, M I, M II, and analyzed the biological information and the cell cycle eggs. The relationship between white D1 and D2 (CCND1 and CCND2) gene and oocyte development was as follows: 1, using the red coloring method to determine the maturation and culture of oocyte from 0 h, 12 h, and 22 h to obtain G V, M I, M II stage oocyte.2, and analyze the sequencing results of the granular cell transcriptional group around three stages of oocyte. It showed that there were 754084917520 transcripts in G V, M I and M II, of which 318 up-regulated genes were up and 304 down-regulated in phase G V and M I, 141 up-regulated and 334 down regulated in phase I and M II stage, 191 up-regulated and 88 down-regulation in G V and M II stage, and qPCR verification results and transcriptional data The results showed that the sequencing data of the transcriptional group were reliable.3, CCND1 and CCND2 were immunofluorescent and immunofluorescent, and the results of real time fluorescence quantitative and Western blot analysis showed that the immunofluorescence of CCND1 and CCND2 protein in granular cells were positive, and the expression level of mRNA and protein in G V granular fine cells was significantly higher than that of M phase I stage. The correlation between M II.4 and the maturation and development of oocyte showed that CCND1 and CCND2 were positively correlated with the rate of in vitro maturation of yak oocytes and the rate of blastocyst after fertilization. In this study, the gene expression profiles of the granulosa cells in Yak oocyte maturation were first obtained by single cell transcriptome. The correlation analysis of the maturation and developmental ability of the parent cells showed that CCND1 and CCND2 were positively related to the developmental potential of oocyte. Thus, it was deduced that CCND1 and CCND2 could be used as biomarkers for evaluating the quality and developmental potential of oocyte, which lay the foundation for the screening of high quality oocyte, the improvement of the mature system in vitro and the development mechanism of yak oocyte. Foundation.

【學(xué)位授予單位】：西南民族大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S823.85

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