【作者】 孟益聪；

【导师】 冯虎元； 邵志峰；

【作者基本信息】 兰州大学 ， 植物学， 2010， 硕士

【摘要】 RNA和染色质之间的联系越来越紧密,这两个领域的交叉点也逐渐被揭示。相比有丝分裂其他时期的染色体形态,中期染色体是迄今可以得到的较大的完整的生物结构,最重要的一点在于其可见的X生物结构。但是到现在还没有关于中期染色体上RNA组成的相关研究,中期染色体由于其结构紧密一般认为是不转录的,那么其上是否存在RNA组分以及它们的组成如何是一个非常有趣的问题,所以这项研究对于中期染色体结构的组成以及表观遗传学都具有重要的指导意义和参考价值。本论文主要是从哺乳动物细胞中分离出完整的中期染色体,提取其上的RNA组分,检测其组成。另外已知一些小RNA分子可以介导异染色质装配以及基因沉默,所以是否在其组分中有一些小的RNA分子存在,它们可能参与中期染色体结构的形成或跟基因表达调控有关。本论文的结果主要有：提取出来的中期染色体RNA主要由核糖体28S,18S和5S亚基组成,同时在用15%的尿素-丙烯酰胺变性胶检测时发现在20-30bp之间有一条比较小的RNA条带出现；建立了中期染色体的提取,中期染色体荧光原位杂交技术,中期染色体RNA提取技术,以及3pmol的少起始量的小分子RNA的克隆等一系列相关技术,从而为下一步分析鉴定中期染色体上小RNA的组成及序列创造了良好的实验基础。更多还原

【Abstract】 In the universe of science, two worlds have recently collided-those of RNA and chromatin. The intersection of these two fields has been impending, but evidence for such a meaningful collision has only recently become apparent.Metaphase chromosome is perhaps the most accessible large scale structural biology project, in comparison to other chromosomal states. The significance is that this is the most visible biological structure (the famous X), yet we virtually have no idea how it is folded and how these can be changes. RNA interference-mediated heterochromation assembly and gene silencing, So far, there is no report on the identity of the RNAs involved. It is an interesting question whether small RNAs are present or required to form the compact structure. It is now shown that heterochromatin may require some transcription that is coupled to small RNA. So this research will provide very important direction and reference value for metaphase chromosome structure and epigenetics. In my paper, I purified metaphase chromosomes from human HT1080 cell line then isolate RNA components from them, see if there’s small RNA fragments that have relationship with metaphase chromosome and transcription regulation exist, and set up a small amount microRNA cloning method, tried to analysis its sequences.Finally I discovered chromosomal RNAs mainly contain ribosomal 28S,18S and 5S subunits, and, I found there is a small band between 20-30bp exsits when detected on 15% polyacrylamide TBE-urea gel.This paper has set up a series of technologies, like metaphase chromosomal isolation method, chromosome in fluorescence situ hybridization, chromosomal RNA isolation method and 3 pmol small initial amount microRNA cloning method by a special molecular cloning procedure based on Weissman’ science paper in 2009, so current results provide a good condition for the future analysis and identification of small RNAs sequences from metaphase chromosome.更多还原