瓯江彩鲤体色变异的转录组与DNA甲基化分析

Genetic regulation mechanism of pigmentation variation has been a major research field of genetics and evolutionary biology in vertebrates. In fish, this research topic has been focused on model fishes, such as zebrafish and medaka fish. However, no reports about genetic regulation mechanism of pigmentation variation has yet been given in common carp. Oujiang color common carp (Cyprinus carpio var. color) has been considered as a good model to conduct fish pigmentation variation study because of its various body color patterns. According to our previously preliminary studies, it was found that black is dominant to red and white, and red is dominant to white, but the genetic mechanism for these observation have not been studied. In this project, first, gene expression difference of transcriptome in skin tissues and DNA methylation in genome will be analyzed for different body color patterns of Oujiang color common carp by using the next-generation sequencing. Second, association analysis between transcriptome and DNA methylation data will be conducted to detect correlations of gene expression and epigenetic modification, and to screen differently expressed genes with methylation regulation. Third, key methylation loci of these genes will be evaluated to test methylation regulation patterns in special DNA sequences by means of bisulfate sequencing. Finally, demethylation experiments will be conducted to detect the association between DNA methylation in special genes and pigmentation variation. This project will provide an insight into genetic regulation mechanism of pigmentation variation in fishes.

体色变异的遗传调控机制是脊椎动物遗传学与进化生物学的重要研究方向。鱼类体色变异的遗传机制研究集中在斑马鱼、青鳉等模式生物，而鲤这方面的研究尚属空白。瓯江彩鲤体色丰富，是研究鱼类体色变异的良好材料。我们前期的表型遗传研究表明：黑色对红色、白色均为显性，红色对白色为显性，但其遗传调控机制尚不清楚。本申请应用第二代测序技术对不同色彩的瓯江彩鲤进行转录组和DNA甲基化分析，了解不同色彩间的基因表达差异与DNA甲基化差异，比较基因表达和表观遗传修饰之间的关系，了解甲基化调控的差异表达基因；应用Bisulfate sequencing技术对差异表达基因的甲基化位点进行验证，判断特定区域DNA甲基化修饰模式；通过DNA去甲基化处理，观察瓯江彩鲤的表观体色变异，研究特定基因甲基化与特定表型之间的关联，解析瓯江彩鲤体色变异的遗传调控机制。

脊椎动物的体色形成及分化是一个复杂的生物学过程，涉及了内在的遗传因子以及外界环境等一系列因素的影响，但相关研究仍比较薄弱。瓯江彩鲤因其丰富的体色和清晰的遗传背景，成为研究鱼类体色形成和进化的重要模式动物。. 本项目以来自于同一家系的4种不同色彩模式的瓯江彩鲤为研究对象，探讨彩鲤体色形成的遗传基础，分别进行了转录组、甲基化组以及功能基因的分析和研究。项目构建了瓯江彩鲤黑、白、红三种体色，四种色彩模式的基因表达谱和甲基化谱，进行了差异表达基因与差异甲基化位点的分析，克隆获得了ASIP和TYRP1等基因的全长序列，利用CRISPR/Case9技术获得了彩鲤ASIP和TYRP1基因敲除的F0代突变体，观察和描述了F0代突变体的体色变异。. 基于上述实验，本项目获得了如下结果：1）比较转录组分析发现瓯江彩鲤黑、红、白三种体色各自具有特异和差异表达的基因，并形成了5种表达模式，特异和差异表达的基因主要集中在黑色素合成和喋啶合成代谢通路，鉴别了多个如ASIP，TYRP1，GPR143，OCA2等与体色形成密切相关的候选基因。2）甲基化组分析的结果描述了4种色彩模式彩鲤的甲基化的富集区域，其中带有黑斑的彩鲤的甲基化程度明显高于纯色的“全红”和“粉玉”。关联分析的结果未发现与与体色基因直接相关的甲基化位点，但是鉴别了如TECR，PPP1R3A等在不同色彩模式彩鲤间存在差异甲基化的与能量代谢相关的基因。3）在前述结果的基础上进行了ASIP和TYRP1基因的功能验证，结果表明ASIP的敲除会导致大花彩鲤背部黑色素细胞零散分布，无法凝集成斑块，推测可能间接参与大花彩鲤黑斑形成，而相反敲除TYRP1基因会造成大花彩鲤黑色素丢失的现象。 . 本项目的研究结果为鱼类乃至脊椎动物的体色形成机制和进化提供了丰富的遗传资源，初步揭示了ASIP和TYRP1基因在彩鲤黑斑形成机制中的遗传调控作用。