基于红腹锦鸡基因组、转录组测序的羽毛色素调控机制研究

发布时间：2018-06-10 09:37

本文选题：基因组 + 转录组　；参考：《内蒙古大学》2016年博士论文

【摘要】：自人类基因组计划启动以来,基因组学逐渐成为研究人员关注的焦点。随着第二代测序技术的诞生,高通量、高深度的基因组测序被用于越来越多的非模式生物。就鸟类而言,2004年鸡基因组测序草图的完成,标志着禽类基因组时代的来临,围绕着鸟类基因组层面的进化关系研究也由此展开。鸟类羽毛颜色丰富多彩,从多种方面赋予鸟类独特而重要的优势,是研究脊椎动物颜色形成的理想模型,因此从鸟类基因组层面研究羽毛色素的调控机制具有极为重要的意义。红腹锦鸡(Chrysolophus pictus)是原产于中国西部的著名观赏雉类。本研究结合红腹锦鸡基因组、转录组测序及羽毛色素成分的分析,对羽毛中黑色素和类胡萝卜素的合成和沉积机理进行了探索。利用二代测序技术,测得了红腹锦鸡全基因组序列和锦鸡羽毛毛囊组织转录组序列,并进行了组装,其中基因组大小为1.029 Gb,注释到16,088个蛋白编码基因,10个样本的转录组组装序列57.66 Gb,注释得到15,118个基因。经过进一步的物种间全基因组比较分析和不同颜色羽毛间的转录组表达分析,以及结合不同颜色羽毛中色素成分的鉴定结果,发现了一系列参与红腹锦鸡羽毛黑色素和类胡萝卜素调控的因子,为开展色素相关基因的功能研究奠定了基础。1.红腹锦鸡与其它鸟类的全基因组比较首先利用由红腹锦鸡基因组序列注释到的单拷贝同源基因构建了系统发育树,表明红腹锦鸡在系统发育地位上与鸡形目中火鸡亲缘关系最近,原鸡次之。然后以斑胸草雀为外群,对鸡、火鸡、红腹锦鸡的全基因组进行了比较分析。在基因拷贝数方面,红腹锦鸡有44个物种特异的基因家族和200个扩张的基因家族,其中的POU家族、涉及脂类代谢的家族和p角蛋白亚家族被认为与黑色素和脂色素的合成和积累有关。在基因共线性方面,红腹锦鸡假染色体中共有58个scaffold基因顺序发生了变化,包括3个大的基因组重排区,受基因组重排影响的基因包括15和黑色素相关基因和24个Hox基因,这些基因直接或间接地参与红腹锦鸡色素的调控。2.红腹锦鸡与白腹锦鸡羽毛色素成分的鉴定利用光谱和色谱的技术对锦鸡不同颜色羽毛的色素成分进行了鉴定。首先进行了拉曼光谱的测定,结果显示红腹锦鸡红、橙、黄色羽毛的羽枝部分和绿色羽毛的羽干部分检测到类胡萝卜素特征峰,而白腹锦鸡红、黄色羽毛显示为褐黑素特征峰,两种锦鸡绿色羽毛为真黑素特征峰。然后对各羽毛进行脂溶性色素的抽提,观察到只有红腹锦鸡的红、橙、黄色羽毛中可以提取到脂溶性色素,将这些物质进行质谱分析并将分子量与数据库比对,发现其中包含分子式为C40H540和C40H5602的类胡萝卜素,其中C40H5602可能是叶黄素和玉米黄素这两种互为同分异构体的化合物。最后进行高效液相色谱的外标法分析,一方面利用叶黄素和玉米黄素标准品分析提取到的类胡萝卜素,另一方面利用PTCA和TTCA分析真黑素和褐黑素,色谱结果与光谱一致,即红腹锦鸡羽毛色素成分包括类胡萝卜素和黑色素,白腹锦鸡羽毛中只有黑色素。3.转录组水平研究锦鸡羽毛色素调控基因分别测定了红腹锦鸡和白腹锦鸡胸腹、颈项、上背、下背、尾羽的羽毛毛囊组织转录组。进行了两个物种同等部位羽毛和不同颜色羽毛的差异表达基因的筛选和聚类分析。对于黑色素相关基因,TYR、 TYRP1、KIT、WNT7在真黑素为主要色素的上背羽毛中表达,ASIP以及溶质载体家族(solute carrier family)的一些成员在褐黑素为主的羽毛中表达。另外,ASIP和MITF由可变剪接产生的转录本中,ASIP-1A和MITF-1M在褐黑素富集的羽毛高表达,推测与褐黑素合成有关。对于类胡萝卜素相关基因,BC02在不含类胡萝卜素的羽毛中表达,参与脂类代谢的基因APOA1和CYP2J主要在含有类胡萝卜素的羽毛中表达,推测可能与类胡萝卜素色素有关。另外,实时定量PCR分析发现,GSTA2和APOD与类胡萝卜素的沉积有关。
[Abstract]:Since the start of the human genome project, genomics has gradually become the focus of researchers. With the birth of the second generation sequencing technology, high throughput, high depth genome sequencing has been used for more and more non pattern organisms. For birds, the completion of the chicken genome sequencing in 2004 marks the advent of the poultry genome era. The study on the evolutionary relationship of the bird genome is also carried out. The color of the bird feathers is rich and colorful and gives birds unique and important advantages. It is an ideal model to study the color formation of vertebrates. Therefore, it is of great significance to study the regulation mechanism of the feather chroma from the bird genome level. Chrysolophus pictus is a famous ornamental pheasant native to Western China. In this study, the synthesis and deposition mechanism of melanin and carotenoid in feathers were explored in combination with the analysis of the genome of Caragana, transcriptional sequence and the composition of feather pigments. The whole genome sequence of the Caragana Caragana was measured by two generation sequencing technology. The sequence of the Caragana feather follicle tissue transcriptional group and the assembly were assembled, in which the size of the genome was 1.029 Gb, annotated to 16088 protein encoding genes, and the transcriptional assembly sequence of 10 samples was 57.66 Gb, and 15118 genes were annotated. After further analysis of the total genome ratio and the transcriptional expression between different colors of different colors. Analysis, and the identification of pigment components in different color feathers, a series of factors involved in the regulation of melanin and carotenoid in Caragana feathers were found, which laid the foundation for the study of the function of the pigments related genes. The whole group of.1. red belly Caragana and other birds first utilized the genome of Caragana Caragana. The sequence annotated single copy homologous genes construct the phylogenetic tree, indicating that the phylogenetic relationship of Caragana in phylogenetic relationship is closest to the chicken in the chicken form, and the original chicken is the next. Then the whole genome of chicken, Turkey and pheasant is compared with the outer group of pectoralis finch. In the number of copies of the gene, there are 44 pheasants in the red belly. The species specific gene family and 200 extended family of genes, the POU family, the family of lipid metabolism and the P keratin family are considered to be related to the synthesis and accumulation of melanin and lipochrome. In the genetic collinearity, the sequence of 58 scaffold genes in the false chromosome of the Caragana pheasant has been changed in sequence, including 3 large Genome rearrangement areas, genes affected by genome rearrangement include 15 and melanin related genes and 24 Hox genes. These genes directly or indirectly participate in the regulation of Caragana coloring in red abdomen,.2. red belly Caragana and white belly Caragana feather pigment composition, using spectrum and chromatography techniques to add pigment composition to different color feathers of Caragana First, the Raman spectrum was measured. The results showed that the Caragana, orange, yellow feather plume and the dry part of the green feather detected the carotenoid characteristic peak, while the white abdomen pheasant red, the Yellow Feather showed the characteristic peak of the brown melanin, and the two kinds of brocade chicken green feathers were the characteristic peak of the melanin. The liposoluble pigments were extracted. Only the red, orange and yellow feathers of Caragana were found to be liposoluble pigments. These substances were analyzed by mass spectrometry and compared with the database. It was found that there were carotenoids with molecular formula of C40H540 and C40H5602, of which C40H5602 may be lutein and zeaxanthin. On the one hand, the carotenoids extracted from the standard products of lutein and zeaxanthin were analyzed by high performance liquid chromatography. On the other hand, the PTCA and TTCA were used to analyze the true melanin and the brown melanin. The chromatographic results and the spectrum are the same, that is, the composition of the red belly Caragana feather pigment includes Hu like Hu. Caragana and melanin, only the melanin.3. transcript level in the Caragana feathers of the Caragana Caragana feathers were studied in the feather follicle tissue of Caragana Caragana and pheasant, neck, upper back, lower back and tail feathers, respectively. The differential expressions of two species of feather and different color feathers were carried out. For melanin related genes, TYR, TYRP1, KIT, WNT7 are expressed in the upper back feathers of the main pigment, and some members of the ASIP and the solute carrier family (solute carrier family) are expressed in the browning main feathers. In addition, ASIP and MITF are produced in the transcriptional transcript of variable splicing, ASIP-1A and MITF. -1M is highly expressed in the feathers enriched with brownish melanin, presumably related to the synthesis of brownish melanin. For carotenoid related genes, BC02 is expressed in the carotenoid free feathers, and the genes involved in lipid metabolism, APOA1 and CYP2J, are mainly expressed in the carotenoid feathers, presumably related to carotenoid pigments. In addition, real time Quantitative PCR analysis revealed that GSTA2 and APOD were related to the deposition of carotenoids.
【学位授予单位】：内蒙古大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q953

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