本文關(guān)鍵詞：基于多組學(xué)數(shù)據(jù)鑒定灘羊被毛卷曲形成相關(guān)基因及其功能分析　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 灘羊卷曲被毛 全基因組甲基化分析 抑制性消減雜交 miRNA-Seq KRT71基因 KRT83基因

【摘要】：灘羊是我國(guó)著名的裘皮用綿羊品種,1月齡灘羊毛發(fā)潔白,卷曲并呈美麗的花穗,俗稱"九道彎",具有很高的經(jīng)濟(jì)價(jià)值,但是"九道彎"特性會(huì)隨著年齡的增長(zhǎng)逐漸消失�；跒┭蜻@一發(fā)育特點(diǎn),本研究采用高通量測(cè)序技術(shù)并整合多組學(xué)數(shù)據(jù)對(duì)上述兩個(gè)生長(zhǎng)階段(1月齡和48月齡)灘羊皮膚組織進(jìn)行分析,獲得與該性狀形成有關(guān)的重要候選基因,并對(duì)其進(jìn)行功能分析,主要獲得了以下初步結(jié)果:1)利用抑制性消減雜交技術(shù)(SSH),在灘羊兩個(gè)生長(zhǎng)階段鑒定出差異表達(dá)基因67個(gè),進(jìn)行GO和KEGG分析發(fā)現(xiàn),差異表達(dá)基因(DGEs)主要富集在細(xì)胞骨架蛋白(BMP)、細(xì)胞粘附分子及其配子等信號(hào)通路。與48月齡相比,1月齡灘羊中高表達(dá)DGEs的4.08%與毛發(fā)生長(zhǎng)相關(guān)。通過(guò)綜合分析,最終獲得20個(gè)(包括KRT71和KRT83)與毛發(fā)生長(zhǎng)相關(guān)基因。2)通過(guò)全基因組重亞硫酸鹽測(cè)序?qū)嶒?yàn)檢測(cè)獲得371million特異性clean reads,覆蓋了整個(gè)參考基因組胞嘧啶的92%以上。結(jié)果顯示得到1,367個(gè)DMRs,占綿羊基因組注釋基因的2.7%。有26個(gè)DMRs落在了基因的啟動(dòng)子區(qū)域,其中5個(gè)相鄰基因(KRT71、CD44、TIFA、ITGBL1和SOX6)直接或者間接與毛發(fā)生長(zhǎng)有關(guān)。3)通過(guò)miR-Seq共鑒定出差異表達(dá)miRNAs有49個(gè)(有12個(gè)已知miRNA參與毛發(fā)生長(zhǎng)),其中在1月齡灘羊中高表達(dá)的有28個(gè),在48月齡灘羊中高表達(dá)的有21個(gè)。對(duì)差異miRNAs的靶基因進(jìn)行GO和KEGG分析,其主要富集到MAPK信號(hào)通路與AMPK信號(hào)通路等與卷曲毛發(fā)生長(zhǎng)相關(guān)的通路。4)KRT71基因啟動(dòng)子活性檢測(cè)實(shí)驗(yàn)和EMSA實(shí)驗(yàn)均發(fā)現(xiàn)KRT71啟動(dòng)子上游-435bp為調(diào)控該基因在灘羊兩個(gè)時(shí)期差異表達(dá)的核心區(qū)域;同時(shí),檢測(cè)到KRT71蛋白在灘羊1月齡組中的表達(dá)量顯著高于48月齡灘羊組。進(jìn)一步DNA甲基化水平檢測(cè)發(fā)現(xiàn)啟動(dòng)子區(qū)(包含-439bp范圍)顯示,48月齡灘羊在該區(qū)域的甲基化水平顯著高于1月齡灘羊,因此推測(cè)KRT71基于啟動(dòng)子區(qū)的DNA甲基化修飾差異可能是造成灘羊兩個(gè)時(shí)期毛發(fā)表型差異的重要原因之。5)KRT83基因是聯(lián)合分析獲得的另一個(gè)影響灘羊被毛卷曲形成的重要基因。通過(guò)細(xì)胞轉(zhuǎn)染實(shí)驗(yàn)和EMSA發(fā)現(xiàn)轉(zhuǎn)錄因子CAP1是調(diào)控KRT83基因mRNA在灘羊兩個(gè)時(shí)期表型差異表達(dá)的重要調(diào)控因子。此外,miRNA干擾實(shí)驗(yàn)發(fā)現(xiàn)oar-miR-432是KRT83蛋白表達(dá)的另一主要調(diào)控因子。基于上述研究結(jié)果,推斷KRT83基因?qū)砬幻纬傻挠绊懯峭ㄟ^(guò)在CAP1和oar-miR-432參與下的轉(zhuǎn)錄前和轉(zhuǎn)錄后調(diào)控來(lái)實(shí)現(xiàn)的。本研究采取多組學(xué)整合分析技術(shù)結(jié)合候選基因的功能分析,系統(tǒng)闡述了灘羊兩個(gè)時(shí)期卷曲被毛表型的差異形成相關(guān)原因。上述研究將為進(jìn)一步揭示灘羊卷曲被毛形成提供新的研究思路和方法。
[Abstract]:Tan is a famous Chinese sheep breeds with fur, wool hair white beach January age, and a beautiful curly panicle, commonly known as the "nine bends", with high economic value, but the "nine bends" characteristics will gradually disappear with age. Tan this development based on the characteristics of this study using high-throughput sequencing technology and integrating multi omics data of the two growth stages (January old and 48 month old) sheep skin tissue were analyzed, and the formation of important traits for some candidate genes, and the functional analysis, get the following preliminary results: 1) suppression subtractive hybridization use (SSH), tan in two growth stages identified 67 differentially expressed genes, were analyzed by GO and KEGG found that the differentially expressed genes (DGEs) are enriched in cytoskeletal protein (BMP), cell adhesion molecules and with signaling pathways. Compared with 48 months of age, January Old Tan sheep high expression in 4.08% and hair growth of DGEs. Through comprehensive analysis, the final 20 (including KRT71 and KRT83) and hair growth related gene.2) 371million specific clean reads by whole genome bisulfite sequencing assay, more than covering the entire reference genome cytosine 92%. The results showed that 1367 DMRs, accounting for sheep genome annotated genes 2.7%. 26 DMRs landed in the promoter regions of genes, in which 5 adjacent genes (KRT71, CD44, TIFA, ITGBL1 and SOX6) directly or indirectly with hair growth of about.3) were identified by miR-Seq expression of miRNAs 49 (there are 12 known miRNA is involved in hair growth), which is highly expressed in January in the old Tan sheep 28, high expression in 48 month old sheep in 21. The analysis of GO and KEGG genes on the difference of miRNAs, its main enrichment to MAPK signal Pathway and AMPK pathway and curly hair growth pathways related to.4) KRT71 gene promoter activity detection experiments and EMSA experiments showed that KRT71 promoter upstream of -435bp gene in the regulation of the core region of Tan sheep two period expression; at the same time, to detect the expression of KRT71 protein in sheep in January age group was significantly higher than that in 48 month old Tan sheep group. Further detection of DNA methylation level found in promoter region (including -439bp range), 48 month old Tan methylation levels in the region was significantly higher than that in January age Tan, suggesting that KRT71 based on DNA methylation differences in promoter region may be an important cause of Tan sheep two period hair phenotype the difference of.5) KRT83 gene is an important gene of another effect of Tan sheep joint analysis is obtained. The formation of hair crimp by cell transfection experiments and EMSA transcription factor CAP1 is the regulation of KRT8 Important factors regulating the expression of 3 mRNA genes in two different period phenotype of Tan sheep. In addition, miRNA interference experiments showed that the oar-miR-432 expression of KRT83 protein is another major regulating factor. Based on the above results, the KRT83 gene is inferred through transcription in CAP1 and oar-miR-432 in the pre - and post transcriptional regulation to achieve the effect of crimp is hair formation. This study adopts multi omics integration analysis technology combined with the function of the candidate gene, introduced sheep two period hair curl difference type table form related reasons. The study will further reveal the tan curly coat formed with new ideas and research methods.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S826

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