四种红藻microRNAs的鉴定与特征分析

发布时间：2018-05-29 08:32

本文选题：红藻 + microRNA　；参考：《山西大学》2016年博士论文

【摘要】：MicroRNAs (miRNAs)是一类广泛存在于真核生物中的内源性非编码小RNAs,其长度约为22nt,有典型的颈环状前体二级结构,通过与靶位点的特异性结合,以切割降解靶mRNA或翻译抑制的方式来调控靶基因的转录后表达。研究表明,miRNAs在调节生物体的生长发育、新陈代谢、细胞分化与凋亡、胁迫应答等方面发挥着重要的作用,是当前生命科学领域研究的热点。红藻是一类地球上进化时间较早、结构相对简单、类群较为庞大、功能多样的藻类。它们不仅具有经济和工业价值,有的还具有医药、保健等多种特殊的生物学功能。尽管目前在动植物中已发现上万条miRNAs,但在低等植物红藻中却鲜有相关报道,对其相关功能的研究则更少。本文选取皱波角叉菜、细齿麒麟菜、紫球藻和温泉红藻四种不同的红藻,运用高通量测序技术联合生物信息学技术首次对四种红藻的miRNAs进行了鉴定和特征分析,这对于进一步充实藻类miRNA数据,弥补四种红藻miRNAs研究的空白,以及对四种红藻特殊功能的深入研究具有十分重要的理论和现实意义。本文的主要研究结果与意义小结如下：1、运用深度测序技术联合生物信息学技术从四种红藻中共鉴定出miRNAs 645条,属于596个miRNA家族,其中保守miRNAs 562条,物种特异miRNAs 83条。2、运用统计学软件和生物信息学技术从数量、长度、碱基偏好性、二级结构、基因家族保守性、靶位点调控方式等方面对四种红藻miRNAs的特征进行了分析。分析结果表明,本研究鉴定的四种红藻miRNAs基本可信。3、用Northern blot和stem-loop qRT-PCR两种不同的实验方法分别对部分红藻miRNAs进行验证。验证分析的结果进一步表明,本文鉴定的四种红藻miRNAs基本可信。4、用psRobot和TargetFinder两种软件在四种红藻miRNAs中共预测到上万条的靶基因和靶位点,psRNATarget软件预测到切割抑制为红藻miRNAs与其靶位点间的主要作用方式,这为将来某些重要miRNA靶基因的表达验证及其调控方式的深入研究奠定了基础。5、基于Gene Ontology平台,我们发现四种红藻中预测的靶基因种类繁多、功能各异,多数可归为生物学过程、分子功能和细胞组分三大类,这也体现了miRNAs功能的多样性。某些潜在的,富集程度较高的,可能在红藻中具有特殊生物学意义的候选靶基因将是今后详细阐明红藻某些重要特征及功能的突破口。6、通过KEGG pathway分析,我们预测到PPAR信号通路、卟啉及叶绿素代谢通路、FcyR介导的吞噬作用和RNA转运通路分别为四种不同红藻靶基因参与调控下的显著富集的代谢通路。这为深入探究各代谢通路,阐明其在红藻生命活动中的重要作用奠定了基础。7、运用Cytoscape软件绘制的miRNA-靶基因互作网络图,以及基于统计学计算下的四种红藻间miRNA数据的比较分析结果,将从整体化和个性化两个角度为红藻miRNAs的深入研究指明方向。此外,miRNA基因家族的进化分析将是未来藻类miRNA研究的新方向。
[Abstract]:MicroRNAs is a class of endogenous non-coding small RNAss widely found in eukaryotes. Its length is about 22nt.It has a typical secondary structure of cervical ring precursor, and it binds specifically to the target site. The post-transcriptional expression of target gene is regulated by cleavage and degradation of target mRNA or translation inhibition. Studies have shown that miRNAs play an important role in regulating the growth and development of organisms, metabolism, cell differentiation and apoptosis, stress response and so on. Red algae is a kind of algae which evolved earlier on the earth, has relatively simple structure, has a large group of species, and has a variety of functions. They not only have economic and industrial value, but also have many special biological functions, such as medicine, health care and so on. Although tens of thousands of miRNAss have been found in plants and animals, there are few reports on the related functions of red algae. In this paper, four different species of red algae, Carrageenia crassifolia, Eucheuma tenuifolia, Chlorella purpurea and red algae from hot springs were selected. The miRNAs of four species of red algae were identified and characterized for the first time by using high-throughput sequencing technique and bioinformatics technique. It is of great theoretical and practical significance to further enrich the miRNA data of algae, to make up for the blank in the study of four species of red algae miRNAs, and to further study the special functions of the four species of red algae. The main results and significance of this study are summarized as follows: 1. A total of 645 miRNAs fragments were identified from four species of red algae by using deep sequencing technique and bioinformatics technique, belonging to 596 miRNA families, among which 596 were conserved miRNAs 562. The characteristics of four species of red algae miRNAs were analyzed by statistical software and bioinformatics from the aspects of quantity, length, base preference, secondary structure, gene family conservatism and target site regulation. The results showed that the miRNAs of four species of red algae identified in this study was basically credible. The Northern blot and stem-loop qRT-PCR methods were used to verify the miRNAs of some red algae. The results of the verification analysis further show that, Four species of red algae miRNAs were identified in this paper. PsRobot and TargetFinder software were used to predict tens of thousands of target genes and target sites in four species of red algae miRNAs. The software predicted that the cleavage inhibition was the main mode of interaction between miRNAs and its target sites. This laid a foundation for the further study of the expression and regulation of some important miRNA target genes in the future. Based on the Gene Ontology platform, we found that the predicted target genes in the four species of red algae have various types and functions, most of which can be classified as biological processes. Molecular function and cell composition are divided into three categories, which also reflect the diversity of miRNAs functions. Some potential, highly enriched candidate target genes, which may have special biological significance in red algae, will be a breakthrough to clarify some important characteristics and functions of red algae in the future. By KEGG pathway analysis, we can predict the PPAR signaling pathway. The phagocytosis mediated by FcyR and the RNA transport pathway by porphyrin and chlorophyll metabolism pathway were four highly enriched metabolic pathways regulated by target genes of red algae respectively. This laid a foundation for further exploring the metabolic pathways and elucidating their important role in the life activities of red algae. The miRNA-target gene interaction network map made by Cytoscape software was used to map the interaction of miRNA-target genes. The results of comparison and analysis of miRNA data among four species of red algae based on statistical calculation will point out the direction of further research on miRNAs from the aspects of integration and individuation. In addition, phylogenetic analysis of miRNA gene family will be a new direction of algal miRNA research in the future.
【学位授予单位】：山西大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

【参考文献】