基于多组学数据鉴定滩羊被毛卷曲形成相关基因及其功能分析

发布时间：2018-01-01 02:26

本文关键词：基于多组学数据鉴定滩羊被毛卷曲形成相关基因及其功能分析　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：滩羊是我国著名的裘皮用绵羊品种,1月龄滩羊毛发洁白,卷曲并呈美丽的花穗,俗称"九道弯",具有很高的经济价值,但是"九道弯"特性会随着年龄的增长逐渐消失。基于滩羊这一发育特点,本研究采用高通量测序技术并整合多组学数据对上述两个生长阶段(1月龄和48月龄)滩羊皮肤组织进行分析,获得与该性状形成有关的重要候选基因,并对其进行功能分析,主要获得了以下初步结果:1)利用抑制性消减杂交技术(SSH),在滩羊两个生长阶段鉴定出差异表达基因67个,进行GO和KEGG分析发现,差异表达基因(DGEs)主要富集在细胞骨架蛋白(BMP)、细胞粘附分子及其配子等信号通路。与48月龄相比,1月龄滩羊中高表达DGEs的4.08%与毛发生长相关。通过综合分析,最终获得20个(包括KRT71和KRT83)与毛发生长相关基因。2)通过全基因组重亚硫酸盐测序实验检测获得371million特异性clean reads,覆盖了整个参考基因组胞嘧啶的92%以上。结果显示得到1,367个DMRs,占绵羊基因组注释基因的2.7%。有26个DMRs落在了基因的启动子区域,其中5个相邻基因(KRT71、CD44、TIFA、ITGBL1和SOX6)直接或者间接与毛发生长有关。3)通过miR-Seq共鉴定出差异表达miRNAs有49个(有12个已知miRNA参与毛发生长),其中在1月龄滩羊中高表达的有28个,在48月龄滩羊中高表达的有21个。对差异miRNAs的靶基因进行GO和KEGG分析,其主要富集到MAPK信号通路与AMPK信号通路等与卷曲毛发生长相关的通路。4)KRT71基因启动子活性检测实验和EMSA实验均发现KRT71启动子上游-435bp为调控该基因在滩羊两个时期差异表达的核心区域;同时,检测到KRT71蛋白在滩羊1月龄组中的表达量显著高于48月龄滩羊组。进一步DNA甲基化水平检测发现启动子区(包含-439bp范围)显示,48月龄滩羊在该区域的甲基化水平显著高于1月龄滩羊,因此推测KRT71基于启动子区的DNA甲基化修饰差异可能是造成滩羊两个时期毛发表型差异的重要原因之。5)KRT83基因是联合分析获得的另一个影响滩羊被毛卷曲形成的重要基因。通过细胞转染实验和EMSA发现转录因子CAP1是调控KRT83基因mRNA在滩羊两个时期表型差异表达的重要调控因子。此外,miRNA干扰实验发现oar-miR-432是KRT83蛋白表达的另一主要调控因子。基于上述研究结果,推断KRT83基因对卷曲被毛形成的影响是通过在CAP1和oar-miR-432参与下的转录前和转录后调控来实现的。本研究采取多组学整合分析技术结合候选基因的功能分析,系统阐述了滩羊两个时期卷曲被毛表型的差异形成相关原因。上述研究将为进一步揭示滩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.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S826

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