山羊胎儿皮肤黑色素生成有关miRNA的筛选与鉴定

发布时间：2018-03-24 04:33

本文选题：山羊　切入点：胎儿　出处：《西南大学》2015年硕士论文

【摘要】：黑色素生成是复杂的生理过程,关于肤色形成的机理已经在人类医学和多数动物上取得了很大的进展,但乌羊的乌皮中乌质性状的形成机理研究相对匮乏。微小RNA (micro-RNA, miRNA)是一类转录后调控基因表达的、长度约为18-25nt的内源性单链RNA,属于非编码小分子RNA。研究报道,miRNAs在皮肤细胞发育、被毛及皮肤颜色控制中起着重要的作用。本研究以皮肤niRNA调控肤色为核心,对乌皮羊及白皮羊胎儿(100日龄)皮肤组织进行小RNA cDNA文库构建及Illumina测序,寻找差异表达的niRNAs。主要研究结果如下：1.通过对山羊皮肤组织小RNA cDNA文库的测序,得到317个符合niRNA二级典型结构特点的序列,其中187个序列是在miRBase v20.0中已报道的山羊(capra hircus)口绵羊(ovis aries)的miRNAs;60个序列是在miRBase v20.0.0中有高度同源的候选niRNAs; 70个序列在niRBase v20.0中无任何同源序列,与上述60个niRNAs合并为本研究新发现的miRNAs。这些新的miRNAs中103个隶属于49个niRNA家族,剩余的miRNA不属于任何家族。对130个新miRNAs序列进行了系统的命名,明确了其在山羊基因组上的座位及正负链上的位置。2.本研究采取三个生物学重复,通过三个比较组对比,最终在乌皮羊和白皮羊100日龄胎儿皮肤中找到了62个差异表达的miRNA,其中31个在乌皮羊胎儿皮肤中表达上调,另外31个在白皮羊胎儿皮肤中高表达。此外,我们选取10个差异表达的1niRNAs对其进行Q-PCR表达验证,随机检测的3个上调miRNA(oar-miR-544, chi-miR-196a和chi-miR-129-5p),7个下调miRNA (oar-miR-133, oar-miR-381, oar-miR-655, oar-miR-543, oar-miR-299, novel-114和chi-miR-184)的Q-PCR表达结果与Ulumina测序分析中得到的结果相符合。3.对62个差异表达的miRNAs进行靶基因的预测及通路显著性富集分析,结果筛选与黑色素生成有关的86个候选靶基因。在这些差异表达miRNAs中, miR-381, miR-543, miR-129在黑色素生成关键基因MITF 3'UTR区域有靶向结合位点；miR-544,miR-129在黑色素生成关键酶TYR 3'UTR区域有靶向结合位点。培养HEK293细胞,双荧光素酶报告系统检测显示,miR-381, miR-543, miR-129与MITF具有靶向作用关系；miR-544, miR-129与TYR有靶向作用关系。同时利用基因本体论GO (Gene Ontology)分类对靶基因功能进行了系统的分类,结果表明这些基因可能参与包括细胞膜成分、分子功能及生物学进程的多个环节。本研究从不同肤色山羊胎儿皮肤中筛选得到了差异表达的miRNAs,并初步发现miR-381, miR-543, miR-129和miR-544可能参与了黑色素生成过程。本研究结果不仅丰富了miRNA数据库中山羊序列的信息,也为山羊胎儿皮肤中黑色素生成机理研究提供了一定的参考数据,同时也为医学研究提供重要的参考价值和意义。
[Abstract]:Melanogenesis is a complex physiological process, and the mechanism of skin color formation has made great progress in human medicine and most animals. However, the formation mechanism of black quality traits in black sheep is relatively scarce. Small RNA micro-RNAs (miRNAs) are a kind of endogenous single-stranded RNAs, which are expressed in posttranscriptional genes and are about the length of 18-25nt, which belong to the non-coding small molecule RNA.The development of miRNAs in skin cells is reported. Hair and skin color control play an important role. In this study, the small RNA cDNA library was constructed and Illumina sequencing was performed on the skin tissue of black sheep and white sheep. The main results of this study were as follows: 1. By sequencing the small RNA cDNA library of goat skin tissue, 317 sequences with typical secondary structure of niRNA were obtained. Of these sequences, 187 were miRNAss in miRBase v20.0, 60 were highly homologous candidate niRNAss in miRBase v20.0.0, and 70 had no homology in niRBase v20.0. In this study, 103 of the new miRNAs belong to 49 niRNA families, and the remaining miRNA does not belong to any family. 130 new miRNAs sequences were systematically named. The loci and positive-negative strands of goat genome were determined. 2. Three biological duplicates were used in this study, and three comparative groups were compared. Finally, 62 differentially expressed miRNAs were found in the 100-day-old fetal skin of black sheep and white sheep, 31 of which were up-regulated in the skin of white sheep and 31 were highly expressed in the skin of white sheep. We selected 10 differentially expressed 1niRNAs to verify their Q-PCR expression. Three up-regulated miRNAs (oar-miR-544, chi-miR-196a and chi-miR-129-5pN), 7 down-regulated Q-PCR expressions of miRNA, oar-miR-131, oar-miR-381, oar-miR-655, oar-miR-543, oar-miR-299, novel-114 and chi-miR-1844) were identified with the results of Ulumina sequencing. Results 86 candidate target genes related to melanogenesis were screened. Among these differentially expressed miRNAs, miR-381, miR-543 and miR-129 have targeted binding sites in the region of MITF 3'UTR, which is the key enzyme of melanogenesis. Target binding sites. HEK293 cells were cultured. The detection of double luciferase report system showed that miR-381, miR-543, miR-129 and MITF have targeting effect relationship, miR-129 and TYR have targeted effect relationship. At the same time, the function of target gene was systematically classified by gene ontology gene ontology classification. The results suggest that these genes may be involved in including cell membrane components, The differential expression of miRNAswas obtained from the fetal skin of goat with different skin color, and it was preliminarily found that miR-381, miR-543, miR-129 and miR-544 may be involved in the process of melanogenesis. The results of this study indicate that miR-381, miR-543, miR-129 and miR-544 may be involved in the process of melanogenesis. It not only enriches the information of goat sequences in miRNA database, It also provides some reference data for the study of melanogenesis mechanism in goat fetal skin, and also provides important reference value and significance for medical research.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827

【参考文献】