青藏高原极端环境下裂腹鱼鳞片退化的分子机制

The Qinghai-Tibet Plateau endemic fish species- Schizothoracinae distributed in the Qinghai-Tibet Plateau and its surrounding areas, in which the uplift of the Qinghai-Tibet Plateau leads to the altitude arising, as well as air density and water temperature decrease dramatically. In the long term of adaptive evolution, the extreme environment stimulated the degradation of Schizothoracinae fishes scales of living at different altitudes. But the molecular mechanism of adaptation to the extreme environment of the Qinghai-Tibet Plateau in Schizothoracinae is unknown. Previous studies showed that ectodysplasin (TNF-related) signaling pathway including EDA, EDAR, and EDARADD, which plays a significant role in the regulating the development of appendages of skin in vertebrate including human and fish. Especially, mutations in EDA and its receptors in fish generated a developmental dysplasia phenotype including osteon deformity and degradation of scales. We had cloned the EDA gene within 20 species of Schizothoracinae at three degradation grade (primitive group, specialized group and highly specialized group) including Gymnocypris, Oxygymnocypris and Diptychus. After sequence alignment and phylogenetic analysis, we found that the evolution of EDA gene supported the degradation of scales in Schizothoracinae. To discover the relationship between EDA signal and Schizothoracinae scale degradation, our project explores the EDA and EDAR gene evolution, function and related pathway via molecular biological and bioinformatics method, gene edit technology. Finally, we provide the evidence that the variation of EDA and EDAR gene involves in the scare degradation of Schizothoracinae, and reveal the molecular mechanism of organism adaption to the extreme environment.

裂腹鱼亚科鱼类分布于青藏高原及其周边地区，随着青藏高原的急剧隆起而起源并演化。高原隆起所导致的海拔、气温、水温等自然条件的改变，对裂腹鱼产生了极大的影响，尤其随着海拔升高、水温降低其在鳞片等表型上发生了明显的退化。然而，目前对于裂腹鱼鳞片退化的分子机制尚不清楚。大量研究已证明以EDA(Ectodysplasin-A)为轴的TNF配体（EDA)-受体(EDAR)-适体(EDARADD)调控通路在脊椎动物的皮肤附件发育中起着至关重要的作用。在鱼上的研究显示该基因及其受体基因的突变、缺失等直接导致骨板、鳞片等的发育异常。申请人前期克隆了不同程度鳞片退化的20种裂腹鱼EDA基因，经序列比对及进化分析证实该基因进化与裂腹鱼鳞片退化相一致。本研究在此基础上拟从探索EDA和EDAR基因变异及调控通路等入手，验证EDA和EDAR基因变异与裂腹鱼鳞片退化的相关性，并探讨EDA信号在鳞片演化中的调控机制。

裂腹鱼亚科鱼类是青藏高原主要的土著鱼类。随着青藏高原的抬升，它们的下咽齿、触须、鳞片等逐渐退化，根据这些特征的退化程度将其分为原始、特化和高度特化三个类群。我们主要从分子水平出发探讨裂腹鱼鳞片退化的分子基础，Ectodysplasin-A (EDA) 在皮肤附件发育中其中主要的调控作用，因此本研究以 EDA为候选基因，采用反转录和over-lap PCR获得裂腹鱼亚科51个种或亚种、鲃亚科5个物种共149个样品的EDA基因的CDS序列。通过序列分析和系统发育树构建，发现EDA基因在一些关键位点发生趋同进化，同时系统发育树结果进一步表明Eda基因的进化与裂腹鱼鳞片的退化呈现一致的趋势，为了进一步验证Eda与裂腹鱼鳞片退化的相关性是特殊的，我们对EDA信号通路中的Edar基因编码区也进行了分析建树，发现该基因进化树与中性基因和Eda都不一致，没有发现与裂腹鱼鳞片退化之间存在相关性。最值得注意的是，与原始类群相比在高度特化类群发现14个SNPs和两个关键缺失（18bp和6bp）；高度特化类群各物种中同时存在的一致的4个非同义突变、和两个G-X-Y 和 1 个XY基序的缺失可能影响EDA蛋白的结构从而影响其功能。硬骨鱼类EDA序列比对显示(G-X-Y)n区域相对保守，高度特化裂腹鱼中存在的两个缺失，并没有在其它鱼类中发现。其外对斑马鱼EDA基因外显子4靶向敲除的实验，证明EDA CL区域与鳞片发育相关。基于以上结果，我们推测Eda 基因的进化可能是裂腹鱼随着海拔升高鳞片逐渐退化的分子基础之一。