山羊痘病毒感染细胞microRNAs的筛

发布时间：2018-05-29 08:47

本文选题：microRNA + 山羊痘病毒　；参考：《中国农业科学院》2015年硕士论文

【摘要】：micro RNAs(mi RNAs)是一类长度约为22nt的非编码小RNA分子,可以通过结合靶基因信使RNA(messager RNA,m RNA)的3'UTR,5'UTR甚至蛋白质编码区域,影响m RNA的稳定性或抑制m RNA的翻译过程,从而调控靶基因所参与的细胞生物学过程。已有多项研究表明宿主细胞mi RNA可以在不同水平上调控感染相关信号通路来调控病毒的生命周期。但是关于mi RNA在痘病毒感染方面的研究较少,本实验以山羊痘病毒(Goatpox virus,GPV)作为研究对象,探究宿主细胞mi RNA在山羊痘病毒感染过程中的作用。本论文采用Illumina/Solexa高通量测序平台对山羊痘病毒侵染的绵羊睾丸(Lamb Testicle,LT)细胞进行mi RNA测序,并对测序数据进行了质量检测和长度分布统计,筛选出差异性表达的mi RNA分子。在此基础上,采用定量PCR方法描述目的mi RNA分子mir-365-3p和mir-365-5p在感染不同时期表达水平变化。通过Target Scan靶基因预测、Gene Onthology(GO)注释分析以及Kyoto Encyclopedia of Genes and Genomes(KEGG)信号通路富集分析,探索mir-365-3p和mir-365-5p在GPV感染过程中可能行使的功能。之后还探究了mir-365-3p和mir-365-5p作为山羊痘病毒感染早期分子诊断标记的可能。主要结果如下:(1)制作LT原代细胞,并测定山羊痘病毒GH株TCID50=10-4.67/100μL。(2)检测了山羊痘病毒感染4h的LT细胞中mi RNA合成途径中关键蛋白Dicer和Argonaute-2(AGO-2)的表达情况,结果发现Dicer蛋白呈上调趋势,而AGO-2表达状态无明显改变。(3)本研究通过Illumina/Solexa高通量测序平台测定了GH株山羊痘病毒感染LT细胞4h后宿主细胞的小RNA转录组,总共鉴定出91个差异性表达的mi RNAs,其中47个为绵羊新发现的mi RNAs。与未感染组相比,这些差异性表达的mi RNAs分子中,有48个mi RNAs下调,43个mi RNAs上调。(4)检测了在山羊痘病毒感染0-48h期间,mir-365-5p和mi-365-3p的表达情况:mir-365-5p主要呈下降趋势,而mir-365-3p则是在0h-10h之间呈上升趋势,10h-48h呈下降趋势。(5)利用Target Scan对mir-365-5p和mir-365-3p的靶基因预测,共得到mir-365-5p的靶基因131个,mir-365-3p的靶基因275个。通过基因本体论GO分析发现mir-365-3p靶基因分子功能主要富集在蛋白质结合,核酸结合以及转录调控因子活性,其生物学过程主要富集在转录调控和细胞基础代谢过程。而mir-365-5p靶基因分子功能主要蛋白质结合,其生物学过程主要富集于蛋白质代谢过程。通过KEGG分析发现mir-365-3p的靶基因信号转导通路显著富集于细胞骨架调控、趋化因子信号通路、Wnt信号通路和丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路,而mir-365-5p的靶基因信号转导通路显著富集于泛素介导的蛋白质降解信号通路和Erb B信号通路。(6)人工接种山羊痘病毒GH株于绵羊肋部表皮和大腿内侧表皮,感染3天后检测肝脏、肺脏、瘤胃和皮肤的mir-365-5p和mir-365-3p的表达情况。结果表明,mir-365-3p在肝脏、肺脏和瘤胃中呈下降趋势,可作为山羊痘病毒感染候选分子诊断标记。
[Abstract]:Micro RNAs (MI RNAs) is a class of non coded small RNA molecules with a length of about 22nt, which can regulate the stability of RNA (messager RNA, m RNA) by combining the target gene messenger RNA (messager RNA, m RNA). The host cell mi RNA can regulate the infection related signal pathway to regulate the life cycle of the virus at different levels. But the research on the MI RNA in the infection of poxvirus is less. This experiment uses the Goatpox virus (GPV) as the research object and explores the role of MI RNA in the process of goat pox virus infection. The MI RNA sequencing of sheep testis (Lamb Testicle, LT) cells infected by goat pox virus was carried out by Illumina/Solexa high throughput sequencing platform. The quality detection and length distribution statistics of the sequenced data were carried out to screen the MI RNA molecules expressed by the travel agents. On this basis, the quantitative PCR method was used to describe the MI RNA molecule mir-365-3p. The expression level of mir-365-5p was changed at different stages of infection. Target Scan target gene prediction, Gene Onthology (GO) annotation analysis and Kyoto Encyclopedia of Genes and Genomes signal pathway enrichment analysis were used to explore the possible functions during the infection process. 365-5p is the possibility of early molecular diagnostic markers for the infection of the goat pox virus. The main results are as follows: (1) the production of LT primary cells and the determination of TCID50=10-4.67/100 mu L. (2) of goat pox virus GH strain were used to detect the expression of the key protein Dicer and Argonaute-2 (AGO-2) in the MI RNA synthesis pathway of 4H infected LT cells. The expression of CER protein was up-regulated, but the expression of AGO-2 was not significantly changed. (3) a small RNA transcriptional group of GH strain LT cell 4H infected LT cells was measured by Illumina/Solexa high throughput sequencing platform. A total of 91 differentially expressed mi RNAs were identified, and 47 of them were newly found in the Mi RNAs. and uninfected groups. Of these differentially expressed mi RNAs molecules, there were 48 mi RNAs downregulation and 43 mi RNAs up-regulated. (4) the expression of mir-365-5p and mi-365-3p was detected during goat pox virus infection 0-48h: mir-365-5p mainly showed a downward trend, while mir-365-3p was in the downward trend between 0h-10h. (5) For the target gene prediction of mir-365-5p and mir-365-3p, 131 target genes of mir-365-5p and 275 target genes of mir-365-3p are obtained. Through the GO analysis of gene ontology, it is found that the function of mir-365-3p target gene is mainly enriched in protein binding, nucleic acid binding and the viability of transcriptional regulators, and the biological process is mainly enriched in the regulation of transcription. And cell based metabolic processes. And mir-365-5p target gene molecular function protein binding, its biological process is mainly enriched in protein metabolism process. Through KEGG analysis, it is found that the target gene signal transduction pathway of mir-365-3p is significantly enriched in cytoskeleton regulation, chemokine signaling pathway, Wnt signaling pathway and mitogen activated protein. Mitogen-activated protein kinase (MAPK) signaling pathway, and mir-365-5p target gene signal transduction pathway is significantly enriched in the ubiquitin mediated protein degradation signaling pathway and Erb B signaling pathway. (6) artificial inoculation of goat pox virus GH strain in the ribs epidermis and the large leg medial epidermis, and detection of the liver, lung, rumen and rumen after 3 days of infection. The expression of mir-365-5p and mir-365-3p in the skin showed that mir-365-3p decreased in the liver, lungs and rumen, and could be used as a diagnostic marker for the candidate molecules of goat pox virus infection.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.654

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