基于单细胞测序技术分析小鼠不同二细胞克隆胚胎转录组差异

发布时间：2018-03-16 22:03

本文选题：单细胞测序　切入点：转录组分析　出处：《阜阳师范学院》2017年硕士论文　论文类型：学位论文

【摘要】：在体细胞核移植领域上最为著名的是Gurdon和Wilmut两人,从Wilmut成功缔造出世界上第一例克隆哺乳动物——克隆绵羊“多莉”以来,极大的促进核移植技术在哺乳动物上应用的研究。尽管到目前为止已有许多种哺乳动物被成功克隆出来,但是从第一只克隆动物的报道距现在已有近59年的历程,当前在高等生物中的核移植效率依然是很低的,大约有1%~3%的出生率。对于核移植胚胎发育率低的科学问题,也因核移植技术的火热而成为热点研究范畴。随后把研究的焦点聚集在克隆胚胎早期发育的机理上。克隆胚胎最为明显的现象之一是早期卵裂阻滞,其发生的时间和胚胎基因组激活的时期一致,在小鼠上是发生在2-cell阶段,其他物种各有差异。基于克隆胚胎发育的问题,本实验室选用B6D2F1杂交小鼠的体内正常胚胎、卵丘细胞核移植胚胎和MEF核移植胚胎三组的2-cell时期的胚胎,作为研究材料,运用单细胞转录组测序技术围绕着2-cell胚胎发育阻滞问题分析转录组数据。通过转录组数据分析得出,筛选出了部分可能影响2-cell胚胎阻滞相关的基因。运用聚类、热图和GO注释等分析方法分析不同胚胎的2-cell时期转录组数据,本实验的测序结果揭示了:在本文中筛选出来的高表达差异基因中,核移植胚胎显著性下调表达这类基因;在筛选出来的三组胚胎中差异表达的基因,核移植胚胎相对于体内正常胚胎,有更多数量的差异表达基因;在表观遗传相关基因、部分OSKM转录因子和细胞周期相关基因的转录组数据分析展示了核移植胚胎与体内正常胚胎之间基因表达水平上差异;对转录组测序中的新转录本基因的分析显示出了,这些新转录本基因在体内正常胚胎普遍为高度活跃的状态,而在核移植胚胎中表达水平较低,且在卵丘细胞核移植胚胎中最不活跃。综上所述,在不同2-cell胚胎转录本中存在显著性差异。比较本实验中我们分析出来的差异表达基因,其中小部分是已有研究报道的,功能是与2-cell阻滞相关的基因,有大部分的基因虽未有在2-cell胚胎发育中功能的研究,我们可以推测这部分基因同样是与2-cell胚胎阻滞紧密相关。用敲低或过表达等实验方法,矫正这些在2-cell时期异常表达的基因可能是提高早期胚胎发育率的一种解决途径。通过对本实验的转录组数据分析,其结果不仅揭示了核移植胚胎与体内正常胚胎转录组之间的差异,同时也为研究核移植胚胎2-cell时期阻滞相关的研究提供了宝贵的线索和指导。
[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.
【学位授予单位】：阜阳师范学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q813;Q78

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