本文選題：單細胞測序　切入點：轉(zhuǎn)錄組分析　出處：《阜陽師范學院》2017年碩士論文　論文類型：學位論文

【摘要】：在體細胞核移植領域上最為著名的是Gurdon和Wilmut兩人,從Wilmut成功締造出世界上第一例克隆哺乳動物——克隆綿羊“多莉”以來,極大的促進核移植技術在哺乳動物上應用的研究。盡管到目前為止已有許多種哺乳動物被成功克隆出來,但是從第一只克隆動物的報道距現(xiàn)在已有近59年的歷程,當前在高等生物中的核移植效率依然是很低的,大約有1%~3%的出生率。對于核移植胚胎發(fā)育率低的科學問題,也因核移植技術的火熱而成為熱點研究范疇。隨后把研究的焦點聚集在克隆胚胎早期發(fā)育的機理上。克隆胚胎最為明顯的現(xiàn)象之一是早期卵裂阻滯,其發(fā)生的時間和胚胎基因組激活的時期一致,在小鼠上是發(fā)生在2-cell階段,其他物種各有差異�；诳寺∨咛グl(fā)育的問題,本實驗室選用B6D2F1雜交小鼠的體內(nèi)正常胚胎、卵丘細胞核移植胚胎和MEF核移植胚胎三組的2-cell時期的胚胎,作為研究材料,運用單細胞轉(zhuǎn)錄組測序技術圍繞著2-cell胚胎發(fā)育阻滯問題分析轉(zhuǎn)錄組數(shù)據(jù)。通過轉(zhuǎn)錄組數(shù)據(jù)分析得出,篩選出了部分可能影響2-cell胚胎阻滯相關的基因。運用聚類、熱圖和GO注釋等分析方法分析不同胚胎的2-cell時期轉(zhuǎn)錄組數(shù)據(jù),本實驗的測序結(jié)果揭示了:在本文中篩選出來的高表達差異基因中,核移植胚胎顯著性下調(diào)表達這類基因;在篩選出來的三組胚胎中差異表達的基因,核移植胚胎相對于體內(nèi)正常胚胎,有更多數(shù)量的差異表達基因;在表觀遺傳相關基因、部分OSKM轉(zhuǎn)錄因子和細胞周期相關基因的轉(zhuǎn)錄組數(shù)據(jù)分析展示了核移植胚胎與體內(nèi)正常胚胎之間基因表達水平上差異;對轉(zhuǎn)錄組測序中的新轉(zhuǎn)錄本基因的分析顯示出了,這些新轉(zhuǎn)錄本基因在體內(nèi)正常胚胎普遍為高度活躍的狀態(tài),而在核移植胚胎中表達水平較低,且在卵丘細胞核移植胚胎中最不活躍。綜上所述,在不同2-cell胚胎轉(zhuǎn)錄本中存在顯著性差異。比較本實驗中我們分析出來的差異表達基因,其中小部分是已有研究報道的,功能是與2-cell阻滯相關的基因,有大部分的基因雖未有在2-cell胚胎發(fā)育中功能的研究,我們可以推測這部分基因同樣是與2-cell胚胎阻滯緊密相關。用敲低或過表達等實驗方法,矯正這些在2-cell時期異常表達的基因可能是提高早期胚胎發(fā)育率的一種解決途徑。通過對本實驗的轉(zhuǎn)錄組數(shù)據(jù)分析,其結(jié)果不僅揭示了核移植胚胎與體內(nèi)正常胚胎轉(zhuǎn)錄組之間的差異,同時也為研究核移植胚胎2-cell時期阻滯相關的研究提供了寶貴的線索和指導。
[Abstract]:Most famous in the field of somatic cell nuclear transfer are Gurdon and Wilmut, since Wilmut successfully created the world's first cloned mammal-cloned sheep "Dolly." It has greatly promoted the application of nuclear transfer technology in mammals. Although many species of mammals have been successfully cloned so far, it has been almost 59 years since the first cloned animal was reported. At present, the efficiency of nuclear transfer in higher organisms is still very low, about one percent of the birth rate. Because of the hot heat of nuclear transfer technology, it has also become a hot research field. Subsequently, the focus of the research focused on the mechanism of the early development of cloned embryos. One of the most obvious phenomena of cloned embryos is early cleavage block. The time of its occurrence is the same as the period of embryonic genome activation. In mice, it occurs in 2-cell stage, and there are differences in other species. Based on the development of cloned embryos, the normal embryos of B6D2F1 hybrid mice were selected in our laboratory. The 2-cell embryos of three groups of cumulus nuclear transfer embryos and MEF nuclear transfer embryos were used as the research materials. The single cell transcriptional sequencing technique was used to analyze the transcriptional data around the 2-cell embryo development retardation. Some genes related to 2-cell embryo block were screened. The transcriptional data of 2-cell period of different embryos were analyzed by cluster, thermal map and go annotation. The sequencing results of this study revealed that nuclear transfer embryos significantly down-regulated the expression of these genes in the highly differentially expressed genes screened in this paper, and differentially expressed genes in the three selected groups of embryos. Nuclear transfer embryos have more differentially expressed genes than normal embryos. The analysis of transcriptional data of some OSKM transcription factors and cell cycle related genes showed the difference in gene expression level between nuclear transfer embryos and normal embryos in vivo, and the analysis of new transcripts in transcriptome sequencing showed that, These new transcripts are generally highly active in normal embryos in vivo, but low in nuclear transfer embryos, and the least active in cumulus nuclear transfer embryos. There are significant differences in different 2-cell embryo transcripts. We compared the differentially expressed genes we analyzed in this experiment, a small number of which have been reported, the function is related to 2-cell block genes. Although most genes have not been studied in 2-cell embryonic development, we can speculate that these genes are also closely related to 2-cell embryo arrest. Correcting the abnormal expression of these genes at 2-cell stage may be a solution to increase the rate of early embryonic development. The results not only reveal the difference between nuclear transfer embryo and normal embryo transcriptional group, but also provide valuable clues and guidance for the study of nuclear transfer embryo 2-cell block.
【學位授予單位】：阜陽師范學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q813;Q78

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