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半滑舌鳎(Cynoglossus semilaevis) 2种视黄酸受体RARα和RARγ克隆及组织表达特性
宋雪松1,2, 徐永江1, 柳学周1, 史宝1, 王滨1, 刘永山1,2, 张雅星1,2
1.青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 中国水产科学研究院黄海水产研究所 青岛 266071;2.上海海洋大学水产与生命学院 上海 201306
摘要:
利用RT-PCR和RACE方法获得了半滑舌鳎(Cynoglossus semilaevis) 2种视黄酸受体RARα和RARγ的cDNA全长序列,并采用定量PCR技术分析了其组织表达特性。半滑舌鳎RARα cDNA序列全长为1823 bp,编码443个氨基酸;RARγ cDNA序列全长为1959 bp,编码489个氨基酸。同源性分析显示,半滑舌鳎RARα和RARγ同源性高达60.8%,与牙鲆(Paralichthys olivaceus)同源性高达97.0%,具有较强的进化保守性。系统进化分析显示,半滑舌鳎RARα和RARγ分别归属于单独的分支,且与其他鱼类聚合成簇。组织表达分析显示,RARα mRNA在肾中表达量最高,而RARγ mRNA在脾中表达量最高,RARα和RARγ mRNA在其他组织中均有表达,表明半滑舌鳎2种RAR都可能参与多种生理过程调控。半滑舌鳎2种RAR mRNA在有眼侧皮肤、眼侧黑化皮肤和眼侧正常皮肤中的表达量依次升高,且发现RARα在正常有眼侧皮肤的表达高于RARγ,而RARγ在眼侧正常皮肤中的表达显著高于RARα,眼侧黑化皮肤中RARα表达高于RARγ。RAR基因在有眼侧和眼侧皮肤组织中的差异表达可能和RA/RAR系统调节体色有关。
关键词:  半滑舌鳎  视黄酸受体  基因克隆  表达模式  眼侧黑化
DOI:
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Molecular Cloning and Spatial Expression of Two Retinoic Acid Receptors RARalpha and RARgamma from Cynoglossus semilaevis
SONG Xuesong1,2, XU Yongjiang1, LIU Xuezhou1, SHI Bao1, WANG Bin1, LIU Yongshan1,2, ZHANG Yaxing1,2
1.Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;2.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306
Abstract:
Two retinoic acid receptors, RARalpha and RARgamma, were cloned from Cynoglossus semilaevis using RT-PCR and RACE methods, and their spatial expression patterns were investigated using a quantitative PCR assay. The results showed that full-length cDNA sequence encoding the RARalpha gene is 1823 bp in length, its open reading frame (ORF) length is 1332 bp, encoding 443 amino acids; the RARgamma cDNA sequence is 1959 bp in length, and the length of ORF is 1497 bp, encoding 489 amino acids. Homology analysis showed that C. semilaevis RARalpha and RARgamma have homology identity of 60.8%, and both have 97% homology identity with the Japanese flounder. Phylogenetic analysis showed that C. semilaevis RARalpha and RARgamma clustered into a separate branch with other fish counterparts. Spatial expression analysis showed that the highest expression level of RARalpha mRNA occurred in the kidney, whereas the highest expression level of RARgamma mRNA was in the spleen. RARgamma was also highly expressed in the gill, kidney, and heart. Furthermore, RARalpha and RARgamma mRNA expression were detected in all examined tissues, which indicated that these two retinoic acid receptors were both involved in multiple physiological regulation processes. In addition, the differential expression of these two RAR genes were found in the blind side and ocular skins, wherein they both had highest expression levels in the normal blind side skin, followed by the blacking blind side skin and had the lowest expression levels in the ocular side skin. In ocular skin and blind-side blacking skin, the RARalpha expression levels were higher than RARgamma but without significant difference, wherein in blind-side normal skin, the RARgamma expressed significantly higher than RARalpha. This differential expression pattern indicated that they might play important physiological roles in blind-side hypermelanosis regulation of Cynoglossus semilaevis.
Key words:  Cynoglossus semilaevis  Retinoic acid receptor  Gene cloning  Expression pattern  Hyperpigmentation