本文關(guān)鍵詞： 東北虎 microRNA 轉(zhuǎn)錄組 靶基因 MAPK信號(hào)通路 表達(dá)調(diào)控　出處：《東北林業(yè)大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：胎盤(pán)是哺乳動(dòng)物妊娠期間由胚胎的胚膜和母體子宮內(nèi)膜聯(lián)合長(zhǎng)成的母子間交換物質(zhì)的過(guò)渡性器官,具有物質(zhì)交換、防御、合成和免疫等功能。隨著東北虎基因組數(shù)據(jù)的公布,東北虎表觀遺傳學(xué)成為一個(gè)可開(kāi)拓的前沿領(lǐng)域。MicroRNA是一類(lèi)由內(nèi)源基因編碼的長(zhǎng)度約為18～25個(gè)核苷酸(nt)的非編碼單鏈RNA分子,其主要參與基因轉(zhuǎn)錄后的表達(dá)調(diào)控。本研究應(yīng)用高通量測(cè)序技術(shù)對(duì)東北虎胎盤(pán)組織進(jìn)行microRNA表達(dá)譜及轉(zhuǎn)錄組分析,并對(duì)胎盤(pán)組織特異microRNA進(jìn)行功能驗(yàn)證,得到以下結(jié)論：1、利用RNA-seq技術(shù)對(duì)東北虎胎盤(pán)組織進(jìn)行small RNA測(cè)序,去除接頭和低質(zhì)量序列后,將clean reads與東北虎基因組(http://www.ncbi.nlm.nih.gov/genome/?term=tiger)進(jìn)行比對(duì)、注釋及表達(dá)分析。獲得東北虎胎盤(pán)組織microRNA表達(dá)譜,其中包括465個(gè)microRNA,經(jīng)聚類(lèi)分析分為41個(gè)簇(cluster)和35個(gè)家族(family),其中胎盤(pán)組織特異性的microRNA有microRNA-424, microRNA-450,microRNA-495, microRNA-376a, microRNA-184, microRNA-369和microRNA-503,利用TAM tools (http://www.cuilab.cn/tam)進(jìn)行功能分析,其功能主要包括胎兒生長(zhǎng)發(fā)育、血管生成、骨再生、肌肉發(fā)育、凋亡與抗凋亡、細(xì)胞增殖與分化、粒細(xì)胞生成、免疫反應(yīng)應(yīng)答和激素分泌調(diào)節(jié)等。2、對(duì)東北虎胎盤(pán)組織轉(zhuǎn)錄組進(jìn)行生物學(xué)分析,包括其表達(dá)基因的Gene Ontology(GO) 和 Clusters of Orthologous Groups of proteins (COG)分析。其中21103條東北虎基因在GO分析中被注釋到45個(gè)GO terms,有8462條基因在COG中被注釋、分類(lèi)。利用靶基因預(yù)測(cè)工具TargetScan和miRanda對(duì)靶基因在東北虎胎盤(pán)組織轉(zhuǎn)錄組上進(jìn)行預(yù)測(cè),獲得東北虎胎盤(pán)組織microRNA調(diào)控靶基因,描述了microRNA靶基因在胎盤(pán)組織中生物學(xué)功能,同時(shí)繪制了microRNA調(diào)控靶基因?qū)崿F(xiàn)胎盤(pán)組織功能的分子網(wǎng)絡(luò)圖。3、選取胎盤(pán)組織中特異的microRNA,利用Real time PCR、組織原位雜交和Westen blotting分析microRNA-424及其靶基因MAP2K1在東北虎和家貓?zhí)ケP(pán)組織中的表達(dá),結(jié)果顯示東北虎胎盤(pán)組織中microRNA-424的表達(dá)顯著低于家貓?zhí)ケP(pán)組織(**p0.01),而其靶基因MAP2K1的表達(dá)在東北虎胎盤(pán)組織中顯著高于家貓?zhí)ケP(pán)組織(**p0.01)。4、通過(guò)靶位點(diǎn)突變及雙熒光素酶分析得出MAP2K13'UTR區(qū)域microRNA-424靶位點(diǎn)突變后,熒光素酶活性無(wú)顯著差異,而未突變組熒光素酶活性顯著下調(diào)(**p0.01),證實(shí)microRNA-424可直接靶向結(jié)合MAP2K13'UTR區(qū)域,并在東北虎成纖維細(xì)胞中過(guò)表達(dá)microRNA-424后,Westen blotting證實(shí)microRNA-424可顯著下調(diào)MAP2K1的蛋白表達(dá)量(**p0.01)。為驗(yàn)證microRNA-424在東北虎成纖維細(xì)胞中的生物學(xué)功能,Westen blotting評(píng)估侵染microRNA-424病毒顆粒后的東北虎成纖維細(xì)胞MAP2K1基因下游ERK1/2的磷酸化及蛋白表達(dá)量,MTT法評(píng)定東北虎成纖維細(xì)胞增值能力,結(jié)果證實(shí),microRNA-424不但直接調(diào)節(jié)MAP2K1的轉(zhuǎn)錄表達(dá),同時(shí)影響下游ERK1/2的表達(dá),從而起到抑制細(xì)胞增殖分化的作用。綜上所述,microRNA-424及其靶基因MAP2K1在東北虎與家貓?zhí)ケP(pán)組織中的表達(dá)差異可能與貓科動(dòng)物體型大小有關(guān),該實(shí)驗(yàn)結(jié)果初步證明東北虎、家貓間的體型大小與microRNA-424的轉(zhuǎn)錄后調(diào)控相關(guān),但具體調(diào)控機(jī)制仍需要在動(dòng)物內(nèi)體進(jìn)行基因敲除或突變實(shí)驗(yàn)來(lái)證實(shí)。
[Abstract]:The placenta is a transitional organ exchange substance during pregnancy by mammalian embryo embryo membrane and the endometrium of a joint between mother and child, with material exchange, defense, synthesis and immune functions. With the published tiger genome data, epigenetic studies as a pioneering frontier is a kind of endogenous encoding by.MicroRNA the gene length is about 18 ~ 25 nucleotides (NT) tiger non encoding single stranded RNA molecules, which are involved in the regulation of the expression of gene transcription. After the application of high-throughput sequencing technology on tiger placenta in microRNA expression profiling and transcriptome analysis, and to verify the function of the placenta specific microRNA. Get the following conclusions: 1. The small RNA sequencing of the Siberian tiger placenta using RNA-seq technology to remove the joint and low quality sequences, clean reads and http://ww group (northeast tiger gene W.ncbi.nlm.nih.gov/genome/? Term=tiger) for comparison, analysis and expression of the notes. Get tiger placenta microRNA expression profiles, including 465 microRNA, cluster analysis was divided into 41 clusters (cluster) and 35 (family), the family of placental tissue specific microRNA microRNA-424, microRNA-450, microRNA-495, microRNA-376a, microRNA-184. MicroRNA-369 and microRNA-503, using TAM tools (http://www.cuilab.cn/tam) functional analysis, its function mainly includes fetal growth, angiogenesis, bone regeneration, muscle development, apoptosis and anti apoptosis, cell proliferation and differentiation, cell formation, immune response and regulation of hormone secretion,.2, biological analysis on tiger placenta the transcriptome, including the gene expression of Gene Ontology (GO) and Clusters of Orthologous Groups of proteins (COG) analysis in 211. 03 tiger genes have been annotated to 45 GO terms in the GO analysis, 8462 genes were annotated in COG, TargetScan and miRanda. Classification prediction tools were used to predict the target gene in the placenta of northeast tiger transcriptome using the target gene, obtained tiger placenta microRNA regulated target genes, is described microRNA biological target gene in placenta, and to draw the molecular network diagram.3 microRNA regulated target genes achieve placenta function, select specific placental tissues by Real microRNA, time PCR, Westen blotting and in situ hybridization analysis of the expression of microRNA-424 and its target gene MAP2K1 in the Siberian tiger and cat in the placenta, the the expression of microRNA-424 in placenta of the tiger cats was significantly lower than that of the placenta (**p0.01), and the expression of its target gene MAP2K1 was significantly higher in the Northeast Tiger placenta In the cat placenta (**p0.01) by.4, the target site mutation and dual luciferase analysis MAP2K13'UTR microRNA-424 target mutation, no significant differences in luciferase activity, while no mutation group significantly reduced luciferase activity (**p0.01), confirmed that microRNA-424 can directly target MAP2K13'UTR region, and over expression of microRNA-424 in fibroblasts in Northeast China tiger, Westen blotting confirmed that microRNA-424 could significantly decrease the protein expression of MAP2K1 (**p0.01). In order to verify microRNA-424 to the biological functions of fibroblasts in the Siberian tiger, the Westen assessment of blotting phosphorylation and protein infection of microRNA-424 virus particles after the tiger into downstream of MAP2K1 gene expression of ERK1/2 cells, MTT evaluation of northeast tiger method of fiber cell proliferation. The results confirmed that the expression of microRNA-424 not only directly regulate the MAP2K1, at the same time. The expression of downstream ERK1/2 ring, thus inhibiting the cell proliferation and differentiation. These differences may be related to the size of the feline animal size of microRNA-424 and the expression of target gene MAP2K1 in the Siberian tiger and cat in the placenta, the experimental results proved that the Siberian tiger, the size and the cat microRNA-424 transcription regulation, but the specific mechanisms still need to be in the animal in gene knockout or mutation experiments to confirm.

【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q953

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張明海;馬建章;;中國(guó)野生東北虎現(xiàn)狀及其保護(hù)愿景展望[J];動(dòng)物學(xué)雜志;2010年01期

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本文編號(hào)：1463825