拟南芥中内含子来源环形RNA的识别与分析

发布时间：2018-01-07 00:15

  本文关键词：拟南芥中内含子来源环形RNA的识别与分析　出处：《昆明理工大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 拟南芥 内含子 分支点 识别 环形RNA

【摘要】：环形RNA(circRNA)是一种结构与常见线性RNA不同的环状的非编码RNA转录物。在生命的各个领域环形RNAs广泛存在,包括真核生物、细菌、古生菌和病毒。最近的研究表明环形RNAs的形成机制和功能具有多样性,例如有外显子环化、内含子环化、细菌基因组环化等。多种形成机制造成了多样性的环形RNAs,具有了多种多样的功能,它能够作为miRNA海绵、RBP海绵、翻译蛋白等。环形RNA以多种形式存在与自然界中,例如真核生物中外显子后向剪接的产物、病毒或类病毒的基因组、古生菌中tRNA和rRNA内含子的剪切产物环形RNAs没有可供核苷酸外切酶识别的3'端,所以能够抵制核苷酸外切酶的降解。稳定、保守,成熟环形RNA在细胞质中大量存在。能够作为miRNAs海绵,典型的例子是miR-7海绵。结合RBP合成RNA-protein合成物,或者调解基因转录。由于环形RNA对RNA酶具有拮抗能力,有较强的稳定性,在分化成熟的细胞中可以缓慢积累,故其可以作为生物分子标记作用于临床、科研。此外有研究表明,环形RNA有可能与microRNA(miRNA)分子、外源性病毒能结合,抑制这些分子的功能。环形RNA可能具有巨大的生物学意义,目前对环形RNA的研究还很欠缺,特别是内含子来源的环形RNA方面。本课题以拟南芥为研究对象,依照环形RNA这类RNA的特点,对内含子分支点、内含子来源的环形RNA进行识别及分析。本课题首先进行实验:核苷酸外切酶(RnaseR酶)处理的实验组及不做任何处理的对照组,共2组4次重复8个样本,然后将试验样本进行高通量测序。根据分支点特点在内含子中识别分支点,并通过分析分支点的保守性、位点等探索其特征。计算8个样本的基因表达水平,通过对基因表达水平进行聚类、离散方式,分析2组样本间的差异。此外通过标准化表达公式对内含子的表达进行计算。依据内含子表达水平筛选内含子来源的环形RNA,选择具有代表性的显著上调的环形RNA。比较基因与内含子在2组的差异,侧重对内含子进行分析。通过长度等特征来对环形RNA进行分析。本课题对环形RNA特征及实验数据进行细致而认真的分析,做出如下成果:1)识别出4080个分支点;2)发现分支点核苷酸具有高度保守性,在3'剪切位点上游19-37nt区域等特征;3)由拟南芥(对照组与实验组)经过RNA酶处理的高通量序列数据,识别出9227个内含子来源环形RNA;4)通过差异性分析找到172个实验组相对对照组显著上调的环形RNA;5)环形RNA长度一般在400bp,比正常内含子长度要更长;综上,本课题分别识别出拟南芥中套索分支点及内含子来源环形RNA,并进一步分析其特征。希望能够为之后拟南芥或环形RNA的研究提供帮助。
[Abstract]:Annular RNAs (rRNAs) are circular non-coding RNA transcripts with different structures from common linear RNA. Circular RNAs exists widely in various fields of life, including eukaryotes. Recent studies have shown that the formation mechanisms and functions of circular RNAs are diverse, such as exon cyclization and intron cyclization. Bacterial genome cyclization and so on. A variety of formation mechanisms led to a variety of ring RNas, with a variety of functions, it can be used as miRNA sponge RBP sponge. Circular RNA exists in many forms with exon backward splicing products in nature, such as eukaryotes, viruses or virus-like genomes. The cleavage product of tRNA and rRNA intron in Archaea has no 3 'end that can be recognized by nucleotide exonuclease, so it can resist the degradation of nucleotide exonuclease and is stable and conservative. Mature ring RNA is abundant in cytoplasm. It can be used as a miRNAs sponge. The typical example is miR-7 sponge. It combines with RBP to synthesize RNA-protein compound. Or mediate gene transcription. Because ring RNA has antagonistic ability to RNA enzyme and has strong stability, it can accumulate slowly in differentiated mature cells, so it can act as biomolecular marker in clinic. In addition, some studies have shown that ring RNA may bind to microRNAs (miRNAs) molecules, foreign viruses can bind. Inhibition of the function of these molecules. Ring RNA may have great biological significance, the current research on circular RNA is still very scarce. Especially the ring RNA of intron origin. In this paper, we take Arabidopsis thaliana as the research object, according to the characteristics of RNA such as ring RNA, branch point of intron. The ring RNA from intron was identified and analyzed. Firstly, the experiment was carried out: the experimental group treated with nucleotide exonuclease (RnaseR) and the control group without any treatment. Eight samples were repeated in 2 groups and 4 times, and then high-throughput sequencing was carried out. According to the characteristics of branch points, branch points were identified in introns, and the conservation of branch points was analyzed. The gene expression level of 8 samples was calculated, and the gene expression level was clustered and discretized. The differences between the two groups were analyzed. In addition, the expression of intron was calculated by standardized expression formula. Ring RNA from intron source was screened according to the expression level of intron. Select the representative significantly up-regulated ring RNA. compare the difference between the gene and intron in the two groups. Focus on the intron analysis. Through length and other characteristics to analyze the annular RNA. This topic on the ring RNA characteristics and experimental data for careful and careful analysis. Make the following result: 1) identify 4080 branch points; 2) it was found that the branched nucleotides were highly conserved, with 19-37 NT region upstream of the 3 'cleavage site. 3) 9227 intron origin annular RNAs were identified from high-throughput sequence data of Arabidopsis thaliana (control group and experimental group) treated with RNA enzyme. 4) based on the difference analysis, 172 experimental groups were found to be significantly up-regulated ring RNAs compared with the control group. 5) the length of annular RNA is generally at 400bp, which is longer than that of normal intron. In summary, we identify the noose branch points and intron origin ring RNAs in Arabidopsis thaliana, and further analyze the characteristics of ring RNAs in Arabidopsis thaliana, and hope to provide some help for the research of Arabidopsis or annular RNA.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2
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本文编号：1390141