本文選題：山羊　切入點：胎兒　出處：《西南大學》2015年碩士論文

【摘要】：黑色素生成是復雜的生理過程,關于膚色形成的機理已經在人類醫(yī)學和多數動物上取得了很大的進展,但烏羊的烏皮中烏質性狀的形成機理研究相對匱乏。微小RNA (micro-RNA, miRNA)是一類轉錄后調控基因表達的、長度約為18-25nt的內源性單鏈RNA,屬于非編碼小分子RNA。研究報道,miRNAs在皮膚細胞發(fā)育、被毛及皮膚顏色控制中起著重要的作用。本研究以皮膚niRNA調控膚色為核心,對烏皮羊及白皮羊胎兒(100日齡)皮膚組織進行小RNA cDNA文庫構建及Illumina測序,尋找差異表達的niRNAs。主要研究結果如下：1.通過對山羊皮膚組織小RNA cDNA文庫的測序,得到317個符合niRNA二級典型結構特點的序列,其中187個序列是在miRBase v20.0中已報道的山羊(capra hircus)口綿羊(ovis aries)的miRNAs;60個序列是在miRBase v20.0.0中有高度同源的候選niRNAs; 70個序列在niRBase v20.0中無任何同源序列,與上述60個niRNAs合并為本研究新發(fā)現的miRNAs。這些新的miRNAs中103個隸屬于49個niRNA家族,剩余的miRNA不屬于任何家族。對130個新miRNAs序列進行了系統(tǒng)的命名,明確了其在山羊基因組上的座位及正負鏈上的位置。2.本研究采取三個生物學重復,通過三個比較組對比,最終在烏皮羊和白皮羊100日齡胎兒皮膚中找到了62個差異表達的miRNA,其中31個在烏皮羊胎兒皮膚中表達上調,另外31個在白皮羊胎兒皮膚中高表達。此外,我們選取10個差異表達的1niRNAs對其進行Q-PCR表達驗證,隨機檢測的3個上調miRNA(oar-miR-544, chi-miR-196a和chi-miR-129-5p),7個下調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表達結果與Ulumina測序分析中得到的結果相符合。3.對62個差異表達的miRNAs進行靶基因的預測及通路顯著性富集分析,結果篩選與黑色素生成有關的86個候選靶基因。在這些差異表達miRNAs中, miR-381, miR-543, miR-129在黑色素生成關鍵基因MITF 3'UTR區(qū)域有靶向結合位點；miR-544,miR-129在黑色素生成關鍵酶TYR 3'UTR區(qū)域有靶向結合位點。培養(yǎng)HEK293細胞,雙熒光素酶報告系統(tǒng)檢測顯示,miR-381, miR-543, miR-129與MITF具有靶向作用關系；miR-544, miR-129與TYR有靶向作用關系。同時利用基因本體論GO (Gene Ontology)分類對靶基因功能進行了系統(tǒng)的分類,結果表明這些基因可能參與包括細胞膜成分、分子功能及生物學進程的多個環(huán)節(jié)。本研究從不同膚色山羊胎兒皮膚中篩選得到了差異表達的miRNAs,并初步發(fā)現miR-381, miR-543, miR-129和miR-544可能參與了黑色素生成過程。本研究結果不僅豐富了miRNA數據庫中山羊序列的信息,也為山羊胎兒皮膚中黑色素生成機理研究提供了一定的參考數據,同時也為醫(yī)學研究提供重要的參考價值和意義。
[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.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S827

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本文編號：1656750