本文選題：鴨腸炎病毒　切入點(diǎn)：miRNA　出處：《四川農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：鴨腸炎病毒(duck enteritis virus, DEV),又稱鴨瘟病毒(duck plague virus, DPV),是一種導(dǎo)致鴨高傳染性、高死亡率的α-皰疹病毒。microRNA(miRNA)為內(nèi)源性的非編碼單鏈小RNA分子,長度約22個(gè)核苷酸,通過與靶基因1nRNA相互作用調(diào)節(jié)基因的表達(dá),在調(diào)節(jié)病毒生活周期、促細(xì)胞存活、免疫逃避和腫瘤發(fā)生等方面發(fā)揮重要作用。鴨腸炎病毒編碼成熟的miRNAs,但對其調(diào)控機(jī)制我們卻未見報(bào)道。本文利用生物信息學(xué)方法預(yù)測了鴨腸炎病毒編碼的33個(gè)niRNAs靶基因,并對dev-miR-D13-5p部分靶基因進(jìn)行了驗(yàn)證及其對病毒增殖的影響進(jìn)行了探討,結(jié)果報(bào)道如下：1. DEV miRNAs靶基因的預(yù)測通過生物信息學(xué)軟件RNAhybrid、miRanda和Targetscan預(yù)測33個(gè)DEV miRNAs的靶基因,得到其可能潛在調(diào)節(jié)40多個(gè)DEV基因和900多個(gè)宿主基因。利用Cytoscape軟件繪制DEV miRNAs和宿主mRNA的互作網(wǎng)絡(luò)。為了更進(jìn)一步探索DEV miRNAs的功能,Ensemble數(shù)據(jù)庫下載宿主靶基因的GO (Gene Ontology)注釋,WEGO生成宿主靶基因GO分類注釋圖,GO分析揭示902個(gè)宿主靶基因映射到42個(gè)GO分類,包括11個(gè)細(xì)胞組分、10個(gè)分子功能和21個(gè)生物學(xué)過程；另外,DAVID分析宿主靶基因的KEGG通路,902個(gè)宿主基因只有一小部分富集于11個(gè)信號(hào)通路,其中MAPK信號(hào)通路富集基因最多。2. dev-miR-D13-5p靶基因的驗(yàn)證為了探究DEV miRNA對病毒增殖的影響,選擇與病毒復(fù)制靶基因UL8(ORF)和UL9 3’UTR緊密相關(guān)的dev-miR-D13-5p為研究對象。本研究首先成功構(gòu)建dev-miR-D13-5p靶基因雙熒光素酶報(bào)告基因野生型載體pmirGLO-UL8(wt)和突變型載體pmirGLO-UL8(mut),然后dev-miR-D13-5p mimic、NC mimic分別與靶基因野生型和突變型載體共轉(zhuǎn)染COS7細(xì)胞,48 h后檢測螢火蟲熒光素酶活性與海腎熒光素酶活性,并計(jì)算兩者熒光素酶活性的比值,結(jié)果野生型試驗(yàn)組(0.3076±0.0289)和對照組(0.4753±0.0497)相比,差異極顯著(P0.01)；突變型試驗(yàn)組(0.4119±0.0147)和對照組(0.4582±0.0435)相比,差異不顯著(P0.05)；野生型試驗(yàn)組(0.3076±0.0289)和突變型試驗(yàn)組(0.4119±0.0147)相比,差異極顯著(P0.01)。表明dev-miR-D13-5p能與UL8(ORF)或UL93'UTR相互作用。3. dev-miR-D13-5p對靶基因轉(zhuǎn)錄水平和病毒增殖的影響構(gòu)建dev-miR-D13-5p靶基因的真核表達(dá)載體pcDNA3.1(+)-UL8(ORF)和pcDNA3.1(+)-UL9(ORF+3'UTR),分別將dev-miR-D13-5p mimic、NC mimic與pcDNA3.1 (+)-UL8(ORF)和pcDNA3.1(+)-UL9(ORF+3'UTR)共轉(zhuǎn)染COS7細(xì)胞,48 h后分別進(jìn)行熒光定量PCR檢測UL8.UL9基因的水平,探討dev-miR-D13-5p mimic對靶基因轉(zhuǎn)錄水平的影響。結(jié)果顯示,與NC mimic組相比,dev-miR-D13-5p mimic組UL8 mRNA的表達(dá)量下調(diào)79.91%,方差分析差異極顯著(P0.01); dev-miR-D13-5p mimic 組 UL9 mRNA的表達(dá)量幾乎無變化,方差分析差異不顯著(P0.05)。據(jù)此表明dev-miR-D13-5p能抑制UL8mRNA水平,但對UL9mRNA水平影響不顯著。為探究dev-miR-D13-5p對病毒增殖的影響,將鴨胚成纖維細(xì)胞(DEF, duck embryo fibroblast)接種至24孔培養(yǎng)板中,一段時(shí)間后轉(zhuǎn)染dev-miR-D13-5p mimic、 NC mimic,轉(zhuǎn)染12h后感染DEV,并于感染DEV后24 h、48 h及72 h后收集細(xì)胞病毒液,絕對熒光定量法檢測DEV DNA的拷貝數(shù)。結(jié)果顯示,24 h和48 h時(shí)dev-miR-D13-5p mimic組(24 h,5.19×104copies/μl; 48h,1.54×105 copies/μl)與NC mimic 組 (24 h,4.8×104copies/μl; 48h,2.28×105copies/μl)相比,差異均不顯著(P0.05)；72h時(shí),dev-miR-D13-5p mimic組(5.86×105copies/μl)顯著低于(P0.05) NC mimic組(2.57×106copies/μl),表明dev-miR-D13-5p能抑制DEV的增殖。
[Abstract]:Duck enteritis virus (duck enteritis, virus, DEV), also known as duck plague virus (duck plague, virus, DPV), is a cause of high mortality of Duck infectious herpes simplex virus.MicroRNA (miRNA) is a non single stranded RNA molecules encoding endogenous, 22 nucleotides that regulate gene expression through the 1nRNA and target gene interactions in the regulation of the viral life cycle, play an important role in promoting cell survival, immune escape and tumor and so on. The duck enteritis virus encoding mature miRNAs, but its mechanism has not been reported. In this paper, we use bioinformatics methods to predict the 33 niRNAs target genes encoding duck enteritis virus dev-miR-D13-5p, and the target genes were validated and its effect on the proliferation of the virus were discussed. The results were reported as follows: 1. DEV prediction of miRNAs target genes by bioinformatics software RNAhybrid, miRanda and T The predicted target genes of 33 DEV miRNAs argetscan, the potential regulation of more than 40 DEV genes and more than 900 genes. The host interaction network drawing DEV miRNAs and host mRNA using Cytoscape software. In order to further explore the function of miRNAs DEV, Ensemble database download host target gene GO (Gene Ontology) notes, WEGO generation the host target gene GO annotation map, GO analysis revealed 902 host target gene mapped to 42 GO classification, including 11 cells, 10 molecular function and 21 biological process; in addition, the DAVID analysis of KEGG pathway of host target gene, 902 host genes enriched in only a small part of the 11 signal the MAPK signaling pathway, gene enrichment at most.2. dev-miR-D13-5p target gene DEV in order to explore the effect of miRNA on the proliferation of the virus, and viral replication target gene UL8 (ORF) and UL9 UTR is close to 3 ' The dev-miR-D13-5p as the research object. Firstly, this study successfully constructed the target gene of dev-miR-D13-5p luciferase reporter vector pmirGLO-UL8 (WT) of wild type and mutant vector pmirGLO-UL8 (MUT), and dev-miR-D13-5p mimic, NC mimic and target gene of wild type and mutant vector were transfected into COS7 cells. After 48 h of firefly luciferase activity detection with the Renilla luciferase activity, and calculate the ratio of luciferase activity, the wild type test group (0.3076 + 0.0289) and control group (0.4753 + 0.0497) compared with significant difference (P0.01); mutation test group (0.4119 + 0.0147) and control group (0.4582 + 0.0435) compared to the difference was not significant (wild type P0.05); experimental group (0.3076 + 0.0289) and mutant test group (0.4119 + 0.0147) compared with significant difference (P0.01). The results indicated that dev-miR-D13-5p and UL8 (ORF) or UL93'UTR.3. dev- interaction Effect of miR-D13-5p on target gene transcription and proliferation of the virus eukaryotic expression vector pcDNA3.1 (+) dev-miR-D13-5p target gene -UL8 (ORF) and pcDNA3.1 (+) -UL9 (ORF+3'UTR) dev-miR-D13-5p mimic NC, respectively, mimic and pcDNA3.1 (+) -UL8 (ORF) and pcDNA3.1 (+) -UL9 (ORF+3'UTR) Co transfection of COS7 cells, UL8.UL9 gene was detected by fluorescent quantitative PCR levels were respectively 48 h, to explore the effects of dev-miR-D13-5p mimic on target gene expression at the transcriptional level. The results showed that, compared with the NC group mimic, expression of dev-miR-D13-5p in group mimic UL8 mRNA down 79.91%, variance analysis (P0.01); no significant changes in expression levels of dev-miR-D13-5p mimic UL9 mRNA almost, variance analysis showed no significant difference (P0.05). It showed that dev-miR-D13-5p can inhibit the level of UL8mRNA, but no significant influence on the level of UL9mRNA. To explore the effect of dev-miR-D13-5p on the proliferation of the virus, will Duck embryo fibroblast (DEF duck, embryo fibroblast) were inoculated into 24 well culture plates, for a period of time after the transfection of dev-miR-D13-5p mimic, NC mimic, DEV infection and infection after transfection of 12h, DEV after 24 h, 48 h and 72 h were collected after the virus liquid, the copy number detection of DEV DNA fluorescence quantitative method. The results showed that mimic group dev-miR-D13-5p 24 h and 48 h (24 h, 5.19 x 104copies/ L; 48h, 1.54 * 105 copies/ L NC) and mimic group (24 h, 4.8 x 104copies/ L; 48h, 2.28 * 105copies/ L) compared to the difference was not significant (P0.05 72h, dev-miR-D13-5p); group mimic (5.86 * 105copies/ L) was significantly lower than that of group mimic (P0.05) NC (2.57 * 106copies/ L), showed that dev-miR-D13-5p could inhibit the proliferation of DEV.

【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65

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相關(guān)期刊論文 前1條

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本文編號(hào)：1620061