鸭肠炎病毒编码microRNAs靶基因的预测及dev-miR-D13-5p对病毒增殖影响的初步研究

发布时间：2018-03-16 13:11

本文选题：鸭肠炎病毒　切入点：miRNA　出处：《四川农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：鸭肠炎病毒(duck enteritis virus, DEV),又称鸭瘟病毒(duck plague virus, DPV),是一种导致鸭高传染性、高死亡率的α-疱疹病毒。microRNA(miRNA)为内源性的非编码单链小RNA分子,长度约22个核苷酸,通过与靶基因1nRNA相互作用调节基因的表达,在调节病毒生活周期、促细胞存活、免疫逃避和肿瘤发生等方面发挥重要作用。鸭肠炎病毒编码成熟的miRNAs,但对其调控机制我们却未见报道。本文利用生物信息学方法预测了鸭肠炎病毒编码的33个niRNAs靶基因,并对dev-miR-D13-5p部分靶基因进行了验证及其对病毒增殖的影响进行了探讨,结果报道如下：1. DEV miRNAs靶基因的预测通过生物信息学软件RNAhybrid、miRanda和Targetscan预测33个DEV miRNAs的靶基因,得到其可能潜在调节40多个DEV基因和900多个宿主基因。利用Cytoscape软件绘制DEV miRNAs和宿主mRNA的互作网络。为了更进一步探索DEV miRNAs的功能,Ensemble数据库下载宿主靶基因的GO (Gene Ontology)注释,WEGO生成宿主靶基因GO分类注释图,GO分析揭示902个宿主靶基因映射到42个GO分类,包括11个细胞组分、10个分子功能和21个生物学过程；另外,DAVID分析宿主靶基因的KEGG通路,902个宿主基因只有一小部分富集于11个信号通路,其中MAPK信号通路富集基因最多。2. dev-miR-D13-5p靶基因的验证为了探究DEV miRNA对病毒增殖的影响,选择与病毒复制靶基因UL8(ORF)和UL9 3’UTR紧密相关的dev-miR-D13-5p为研究对象。本研究首先成功构建dev-miR-D13-5p靶基因双荧光素酶报告基因野生型载体pmirGLO-UL8(wt)和突变型载体pmirGLO-UL8(mut),然后dev-miR-D13-5p mimic、NC mimic分别与靶基因野生型和突变型载体共转染COS7细胞,48 h后检测萤火虫荧光素酶活性与海肾荧光素酶活性,并计算两者荧光素酶活性的比值,结果野生型试验组(0.3076±0.0289)和对照组(0.4753±0.0497)相比,差异极显著(P0.01)；突变型试验组(0.4119±0.0147)和对照组(0.4582±0.0435)相比,差异不显著(P0.05)；野生型试验组(0.3076±0.0289)和突变型试验组(0.4119±0.0147)相比,差异极显著(P0.01)。表明dev-miR-D13-5p能与UL8(ORF)或UL93'UTR相互作用。3. dev-miR-D13-5p对靶基因转录水平和病毒增殖的影响构建dev-miR-D13-5p靶基因的真核表达载体pcDNA3.1(+)-UL8(ORF)和pcDNA3.1(+)-UL9(ORF+3'UTR),分别将dev-miR-D13-5p mimic、NC mimic与pcDNA3.1 (+)-UL8(ORF)和pcDNA3.1(+)-UL9(ORF+3'UTR)共转染COS7细胞,48 h后分别进行荧光定量PCR检测UL8.UL9基因的水平,探讨dev-miR-D13-5p mimic对靶基因转录水平的影响。结果显示,与NC mimic组相比,dev-miR-D13-5p mimic组UL8 mRNA的表达量下调79.91%,方差分析差异极显著(P0.01); dev-miR-D13-5p mimic 组 UL9 mRNA的表达量几乎无变化,方差分析差异不显著(P0.05)。据此表明dev-miR-D13-5p能抑制UL8mRNA水平,但对UL9mRNA水平影响不显著。为探究dev-miR-D13-5p对病毒增殖的影响,将鸭胚成纤维细胞(DEF, duck embryo fibroblast)接种至24孔培养板中,一段时间后转染dev-miR-D13-5p mimic、 NC mimic,转染12h后感染DEV,并于感染DEV后24 h、48 h及72 h后收集细胞病毒液,绝对荧光定量法检测DEV DNA的拷贝数。结果显示,24 h和48 h时dev-miR-D13-5p mimic组(24 h,5.19×104copies/μl; 48h,1.54×105 copies/μl)与NC mimic 组 (24 h,4.8×104copies/μl; 48h,2.28×105copies/μl)相比,差异均不显著(P0.05)；72h时,dev-miR-D13-5p mimic组(5.86×105copies/μl)显著低于(P0.05) NC mimic组(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.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

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