本文選題：microRNA + 山羊痘病毒�。� 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：micro RNAs(mi RNAs)是一類長(zhǎng)度約為22nt的非編碼小RNA分子,可以通過結(jié)合靶基因信使RNA(messager RNA,m RNA)的3'UTR,5'UTR甚至蛋白質(zhì)編碼區(qū)域,影響m RNA的穩(wěn)定性或抑制m RNA的翻譯過程,從而調(diào)控靶基因所參與的細(xì)胞生物學(xué)過程。已有多項(xiàng)研究表明宿主細(xì)胞mi RNA可以在不同水平上調(diào)控感染相關(guān)信號(hào)通路來調(diào)控病毒的生命周期。但是關(guān)于mi RNA在痘病毒感染方面的研究較少,本實(shí)驗(yàn)以山羊痘病毒(Goatpox virus,GPV)作為研究對(duì)象,探究宿主細(xì)胞mi RNA在山羊痘病毒感染過程中的作用。本論文采用Illumina/Solexa高通量測(cè)序平臺(tái)對(duì)山羊痘病毒侵染的綿羊睪丸(Lamb Testicle,LT)細(xì)胞進(jìn)行mi RNA測(cè)序,并對(duì)測(cè)序數(shù)據(jù)進(jìn)行了質(zhì)量檢測(cè)和長(zhǎng)度分布統(tǒng)計(jì),篩選出差異性表達(dá)的mi RNA分子。在此基礎(chǔ)上,采用定量PCR方法描述目的mi RNA分子mir-365-3p和mir-365-5p在感染不同時(shí)期表達(dá)水平變化。通過Target Scan靶基因預(yù)測(cè)、Gene Onthology(GO)注釋分析以及Kyoto Encyclopedia of Genes and Genomes(KEGG)信號(hào)通路富集分析,探索mir-365-3p和mir-365-5p在GPV感染過程中可能行使的功能。之后還探究了mir-365-3p和mir-365-5p作為山羊痘病毒感染早期分子診斷標(biāo)記的可能。主要結(jié)果如下:(1)制作LT原代細(xì)胞,并測(cè)定山羊痘病毒GH株TCID50=10-4.67/100μL。(2)檢測(cè)了山羊痘病毒感染4h的LT細(xì)胞中mi RNA合成途徑中關(guān)鍵蛋白Dicer和Argonaute-2(AGO-2)的表達(dá)情況,結(jié)果發(fā)現(xiàn)Dicer蛋白呈上調(diào)趨勢(shì),而AGO-2表達(dá)狀態(tài)無(wú)明顯改變。(3)本研究通過Illumina/Solexa高通量測(cè)序平臺(tái)測(cè)定了GH株山羊痘病毒感染LT細(xì)胞4h后宿主細(xì)胞的小RNA轉(zhuǎn)錄組,總共鑒定出91個(gè)差異性表達(dá)的mi RNAs,其中47個(gè)為綿羊新發(fā)現(xiàn)的mi RNAs。與未感染組相比,這些差異性表達(dá)的mi RNAs分子中,有48個(gè)mi RNAs下調(diào),43個(gè)mi RNAs上調(diào)。(4)檢測(cè)了在山羊痘病毒感染0-48h期間,mir-365-5p和mi-365-3p的表達(dá)情況:mir-365-5p主要呈下降趨勢(shì),而mir-365-3p則是在0h-10h之間呈上升趨勢(shì),10h-48h呈下降趨勢(shì)。(5)利用Target Scan對(duì)mir-365-5p和mir-365-3p的靶基因預(yù)測(cè),共得到mir-365-5p的靶基因131個(gè),mir-365-3p的靶基因275個(gè)。通過基因本體論GO分析發(fā)現(xiàn)mir-365-3p靶基因分子功能主要富集在蛋白質(zhì)結(jié)合,核酸結(jié)合以及轉(zhuǎn)錄調(diào)控因子活性,其生物學(xué)過程主要富集在轉(zhuǎn)錄調(diào)控和細(xì)胞基礎(chǔ)代謝過程。而mir-365-5p靶基因分子功能主要蛋白質(zhì)結(jié)合,其生物學(xué)過程主要富集于蛋白質(zhì)代謝過程。通過KEGG分析發(fā)現(xiàn)mir-365-3p的靶基因信號(hào)轉(zhuǎn)導(dǎo)通路顯著富集于細(xì)胞骨架調(diào)控、趨化因子信號(hào)通路、Wnt信號(hào)通路和絲裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信號(hào)通路,而mir-365-5p的靶基因信號(hào)轉(zhuǎn)導(dǎo)通路顯著富集于泛素介導(dǎo)的蛋白質(zhì)降解信號(hào)通路和Erb B信號(hào)通路。(6)人工接種山羊痘病毒GH株于綿羊肋部表皮和大腿內(nèi)側(cè)表皮,感染3天后檢測(cè)肝臟、肺臟、瘤胃和皮膚的mir-365-5p和mir-365-3p的表達(dá)情況。結(jié)果表明,mir-365-3p在肝臟、肺臟和瘤胃中呈下降趨勢(shì),可作為山羊痘病毒感染候選分子診斷標(biāo)記。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.654

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相關(guān)期刊論文 前1條

1 陳軼霞;才學(xué)鵬;;羊痘病毒分子特征及檢測(cè)方法研究進(jìn)展[J];畜牧與獸醫(yī);2008年11期

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本文編號(hào)：1950256