發(fā)布時(shí)間：2018-03-24 04:33

  本文選題：山羊　切入點(diǎn)：胎兒　出處：《西南大學(xué)》2015年碩士論文

【摘要】：黑色素生成是復(fù)雜的生理過(guò)程,關(guān)于膚色形成的機(jī)理已經(jīng)在人類(lèi)醫(yī)學(xué)和多數(shù)動(dòng)物上取得了很大的進(jìn)展,但烏羊的烏皮中烏質(zhì)性狀的形成機(jī)理研究相對(duì)匱乏。微小RNA (micro-RNA, miRNA)是一類(lèi)轉(zhuǎn)錄后調(diào)控基因表達(dá)的、長(zhǎng)度約為18-25nt的內(nèi)源性單鏈RNA,屬于非編碼小分子RNA。研究報(bào)道,miRNAs在皮膚細(xì)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(diào)控膚色為核心,對(duì)烏皮羊及白皮羊胎兒(100日齡)皮膚組織進(jìn)行小RNA cDNA文庫(kù)構(gòu)建及Illumina測(cè)序,尋找差異表達(dá)的niRNAs。主要研究結(jié)果如下：1.通過(guò)對(duì)山羊皮膚組織小RNA cDNA文庫(kù)的測(cè)序,得到317個(gè)符合niRNA二級(jí)典型結(jié)構(gòu)特點(diǎn)的序列,其中187個(gè)序列是在miRBase v20.0中已報(bào)道的山羊(capra hircus)口綿羊(ovis aries)的miRNAs;60個(gè)序列是在miRBase v20.0.0中有高度同源的候選niRNAs; 70個(gè)序列在niRBase v20.0中無(wú)任何同源序列,與上述60個(gè)niRNAs合并為本研究新發(fā)現(xiàn)的miRNAs。這些新的miRNAs中103個(gè)隸屬于49個(gè)niRNA家族,剩余的miRNA不屬于任何家族。對(duì)130個(gè)新miRNAs序列進(jìn)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(fù)鏈上的位置。2.本研究采取三個(gè)生物學(xué)重復(fù),通過(guò)三個(gè)比較組對(duì)比,最終在烏皮羊和白皮羊100日齡胎兒皮膚中找到了62個(gè)差異表達(dá)的miRNA,其中31個(gè)在烏皮羊胎兒皮膚中表達(dá)上調(diào),另外31個(gè)在白皮羊胎兒皮膚中高表達(dá)。此外,我們選取10個(gè)差異表達(dá)的1niRNAs對(duì)其進(jìn)行Q-PCR表達(dá)驗(yàn)證,隨機(jī)檢測(cè)的3個(gè)上調(diào)miRNA(oar-miR-544, chi-miR-196a和chi-miR-129-5p),7個(gè)下調(diào)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(dá)結(jié)果與Ulumina測(cè)序分析中得到的結(jié)果相符合。3.對(duì)62個(gè)差異表達(dá)的miRNAs進(jìn)行靶基因的預(yù)測(cè)及通路顯著性富集分析,結(jié)果篩選與黑色素生成有關(guān)的86個(gè)候選靶基因。在這些差異表達(dá)miRNAs中, miR-381, miR-543, miR-129在黑色素生成關(guān)鍵基因MITF 3'UTR區(qū)域有靶向結(jié)合位點(diǎn)；miR-544,miR-129在黑色素生成關(guān)鍵酶TYR 3'UTR區(qū)域有靶向結(jié)合位點(diǎn)。培養(yǎng)HEK293細(xì)胞,雙熒光素酶報(bào)告系統(tǒng)檢測(cè)顯示,miR-381, miR-543, miR-129與MITF具有靶向作用關(guān)系；miR-544, miR-129與TYR有靶向作用關(guān)系。同時(shí)利用基因本體論GO (Gene Ontology)分類(lèi)對(duì)靶基因功能進(jìn)行了系統(tǒng)的分類(lèi),結(jié)果表明這些基因可能參與包括細(xì)胞膜成分、分子功能及生物學(xué)進(jìn)程的多個(gè)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(dá)的miRNAs,并初步發(fā)現(xiàn)miR-381, miR-543, miR-129和miR-544可能參與了黑色素生成過(guò)程。本研究結(jié)果不僅豐富了miRNA數(shù)據(jù)庫(kù)中山羊序列的信息,也為山羊胎兒皮膚中黑色素生成機(jī)理研究提供了一定的參考數(shù)據(jù),同時(shí)也為醫(yī)學(xué)研究提供重要的參考價(jià)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(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S827

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