红鳍笛鲷皮肤转录组分析及体色相关基因克

发布时间：2018-04-15 20:47

本文选题：红鳍笛鲷 + RNA-seq　；参考：《湖南师范大学》2016年博士论文

【摘要】：动物体色在其生命过程中发挥着重要作用,从生物识别到生态交流甚至与物种分化都有关系。虽然制约动物体色表现的因素多种多样,但由遗传决定的色素细胞发育和分布仍然是决定体色形式的主要因子。红鳍笛鲷是我国南海重要的经济鱼类,体色鲜艳,主体呈现红色具有一定的观赏性,体色是决定其经济价值的重要指标之一。但近年来,人工养殖红鳍笛鲷逐渐出现体色变淡或变黑的现象,影响其经济价值。同时笛鲷属鱼类属于典型的岩礁鱼类,属内物种体色多样,前期研究推测体色与成种具有一定的相关性。因此,在本研究中,首先对红鳍笛鲷个体发育中体色形成进行了跟踪,通过显微和透射电镜技术对红鳍笛鲷黑色和红色皮肤色素细胞的种类和分布情况进行了观察;利用高通量测序技术,进一步研究了红鳍笛鲷黑色和红色皮肤部位基因存在和表达情况,通过比较不同颜色转录组中差异表达基因,发掘与体色形成有关的基因和通路;并克隆了红鳍笛鲷中与黑色体色有关的mitf和酪氨酸酶基因家族基因,研究了它们在红鳍笛鲷中的复制基因的存在情况、在个体发育与成体不同组织中的表达情况。大量体色基因的发掘为后续功能基因研究的开展和分子育种工作的进行以及笛鲷属鱼类体色与进化关系的分析提供有价值的信息。体色有关功能基因在红鳍笛鲷中复制基因存在情况和表达模式的研究,为进一步研究复制基因在红鳍笛鲷中的功能分化提供基础。主要研究结果如下:1.红鳍笛鲷个体发育过程中首先出现黑色素细胞,至1dhf躯干部顶端才开始有微弱黄色素细胞出现。显微观察发现红鳍笛鲷成体皮肤中色素细胞主要分布在真皮层。透射电镜观察发现红鳍笛鲷皮肤中含有黑色素细胞、红色素细胞、虹彩色素细胞和黄色素细胞四种类型。四种色素细胞在黑色和红色皮肤中都有分布,颜色差异主要是由色素细胞类型和数量不同造成的,黑色皮肤中以黑色素细胞数量居多,红色皮肤部位则多为红色素细胞。2.利用高通量测序技术分别获得了红鳍笛鲷黑色和红色皮肤转录组中49,531,098和51,438,110条clean reads。通过序列拼接分别得到了142,792和122,508条Unigenes在红鳍笛鲷黑色和红色皮肤中表达。经数据库注释分别得到50,220和49,736条注释Unigenes。KEGG和COG功能注释发现两种颜色皮肤中表达基因在功能分类上并没有明显差别。3.通过对红鳍笛鲷黑色和红色皮肤中基因表达量分析,共发现9,200个差异表达Unigenes。GO分类表明一部分与细胞结构和酶催化活性有关的基因在两种皮肤中差异表达。KEGG分析发现,与糖酵解、氧化磷酸化、柠檬酸循环和蛋白酶体有关的通路中有较多的基因参与了红色皮肤的形成;酪氨酸酶代谢和黑色素生成通路中有较多的差异表达基因参与了黑色皮肤的形成。关于这些通路中基因的差异表达是否直接与体色形成有关,还需要进一步验证。此外,通过与已知斑马鱼体色基因的比对,在红鳍笛鲷中发现87个匹配基因,部分基因在红鳍笛鲷中存在两个复制基因。其中slc45a2、pmela等基因在黑色皮肤中显著高表达,arl6、aldoaa在红色皮肤中显著高表达。4.结合转录组数据,对黑色素细胞分化、发育和黑色素生成过程中的关键基因mitf进行了克隆。发现红鳍笛鲷中存在mitfa和mitfb两个基因,分别为1,743bp,编码408个氨基酸和1,866bp,编码462个氨基酸。结构预测发现转录因子bHLH家族特有结构域在两个基因中高度保守。基因表达分析推测两个基因功能存在分化,mitfb较多保留mitf基因功能。mitfb在个体发育过程和成体中均有表达,mitfa在个体发育过程和成体表达均弱于mitfb。荧光定量PCR分析发现mitfb在成体眼睛和黑色皮肤部位的表达量显著高于mitfa。5.红鳍笛鲷中酪氨酸酶基因家族包含tyr、tyrp1a、tyrp1b和tyrp2基因。在红鳍笛鲷中,4个基因在黑色素合成部位均显示最高表达,表明其功能保守性。分析tyrp1a和tyrp1b功能分化,推测tyrp1b较多保留了tyrp1基因的功能。
[Abstract]:Animal body color plays an important role in the process of life, from biological recognition to ecological and species differentiation exchange or even have a relationship. Although many factors showed the diversity of animal body color, but the pigment cells genetically determined the distribution and development is still the major factors determining the color form. Red snapper is important in the South China Sea the main economic fish, brightly colored, red has certain ornamental, color is one of the important indicators to determine its economic value. But in recent years, artificial breeding of red snapper gradually fades color or black phenomenon, affecting its economic value. At the same time, snappers belong to typical reef fish species. Color species diversity, previous research that has a certain correlation with the color of kind. Therefore, in this study, firstly lutjanuserythopterus ontogeny of color formation of tracking, through explicit The variety and distribution of micro and transmission electron microscopy of lutjanuserythopterus black and red skin pigment cells were observed; the use of high-throughput sequencing technology, further study of the gene of lutjanuserythopterus black and red skin and the expression of gene expression, by comparing the different colors of the transcriptome, and explore the color formation and gene pathway; cloning associated with black color MITF and tyrosinase gene lutjanuserythopterus, studies the existing situation in lutjanuserythopterus replication in gene expression, in individual development and in adult tissues. A large number of genes for exploring color provides valuable development and Molecular Breeding Research on gene function of the follow-up and analysis of snappers bodycolor and evolutionary relationship information. Color related function gene in lutjanuserythopterus complex Business models and research of gene expression, and provide the basis for further study on the functional differentiation of gene duplication in the red snapper. The main results are as follows: first melanocytes 1. lutjanuserythopterus during ontogeny, to 1dhf trunk top began to have weak yellow pigment cells. Microscopic observation found red snapper adult skin pigment cells were mainly distributed in the dermis. Transmission electron microscopy found in melanoma cells, red snapper skin pigment cells, iris pigment cells and yellow pigment cells. Four types of four kinds of pigment cells are distributed in black and red skin, color difference is mainly caused by the pigment cell type and the number of different, black skin in the number of melanoma cells are, red skin is more pigment cells by.2. high-throughput sequencing were obtained from red Black and red snapper skin transcriptome in 49531098 and 51438110 clean reads. sequence were obtained by expression of 142792 and 122508 Unigenes in lutjanuserythopterus black and red skin. The notes were obtained 50220 and 49736 database notes Unigenes.KEGG and COG can find notes two colors in the skin of gene expression in a functional classification there is no significant difference of.3. through the analysis of the gene expression of lutjanuserythopterus black and red skin, Unigenes.GO classification showed catalytic activity with a portion of the cell structure and enzyme related genes in two kinds of skin.KEGG expression analysis found 9200 differentially expressed were found, and glycolysis, oxidative phosphorylation, lemon gene acid cycle and proteasome associated with more involved in the pathway of formation of red skin; melanin and tyrosinase metabolism pathway There are many differences between the expression of genes involved in the formation of black skin. Whether directly related to the formation and color difference of gene expression in these pathways, need to be further verified. In addition, by comparing with known genes in zebrafish color, 87 matching gene found in red snapper, part of the gene replication gene in two red snapper. The SLC45A2, pmela gene in black skin tissues, arl6, aldoaa in red skin significantly high expression of.4. binding transcription group data on melanocyte differentiation, development and production of melanin in the process of key gene MITF were cloned. The discovery of the existence of mitfa and mitfb two the gene of lutjanuserythopterus, respectively 1743bp, encoding 408 amino acids and 1866bp, encoding 462 amino acids. The structure prediction showed that the bHLH transcription factor family specific domain highly conserved among the two genes. For analysis of the expression of two genes that function in differentiation, more mitfb retain MITF.Mitfb gene function in individual development and expressed in the body, mitfa in the process of individual development and adult expression was weaker than that of mitfb. fluorescent quantitative PCR analysis showed that the expression of mitfb is significantly higher than those in adult eyes and black skin tyrosinase mitfa.5. red snapper gene family contains Tyr, tyrp1a, tyrp1b and TYRP2 genes. In lutjanuserythopterus, 4 genes showed the highest expression in melanin synthesis site, show that its function is conservative. Analysis of tyrp1a and tyrp1b can retain more differentiation, suggesting that tyrp1b TYRP1 gene function.

【学位授予单位】：湖南师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4

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