金钱鱼Dmrt4基因的克隆及表达分析

Molecular Cloning and Expression Profile of Dmrt4 in Spotted Scat(Scatophagus argus)

【目的】研究金钱鱼(Scatophagus argus)Dmrt4(Doublesex and Mab-3 related transcription factors 4)基因在性腺和胚胎发育中的表达。【方法】克隆金钱鱼Dmrt4,采用逆转录PCR检测Dmrt4的组织表达,用实时荧光定量PCR检测Dmrt4在性腺和胚胎发育过程中的表达。【结果与结论】金钱鱼Dmrt4的开放阅读框(ORF)全长1 260 bp,编码蛋白全序列为419个氨基酸。金钱鱼Dmrt4氨基酸序列含有DM和DMA结构域。金钱鱼Dmrt4与欧洲海鲈(Dicentrarchus labrax)和大黄鱼(Larimichthys crocea)的同源性最高,为87.6%;与非洲爪蟾(Xenopus laevis)的同源性最低,为47.1%。金钱鱼Dmrt4与其他鲈形目鱼类在系统进化树上聚为一支。金钱鱼Dmrt4在精巢、卵巢和鳃中高表达,在Ⅲ和Ⅳ期精巢中的表达显著高于V期精巢和Ⅱ、Ⅲ和Ⅳ期卵巢。胚胎发育过程中Dmrt4均有表达,原肠胚的表达水平最高。

【Objective】To evaluate the Dmrt(Doublesex and Mab-3 related transcription factors)gene family which is important for the regulation of reproduction and development in animals.However,the information of Dmrt4 in Scatophagus argus is lacking.【Method】The Dmrt4 gene of S.argus is cloned,and its tissue distribution and expression profiles at different gonad and embryo developmental stages were analyzed by reverse transcription PCR(RT-PCR)and real-time quantitative PCR(qPCR).【Result and Conclusion】The spotted scat Dmrt4 full-length ORF is 1260 bp and encodes 419 amino acids.Sequence analysis revealed that Dmrt4 consists of DM and DMA domains.Multiple sequence alignment showed that S.argus Dmrt4 was highly homologous to Dmrt4 from Dicentrarchus labrax and Larimichthys crocea(87.6%),but it was less homologous to frog Xenopus laevis(47.1%)at amino acid level.Phylogenetic analysis revealed that Dmrt4 is closest to D.labrax and L.crocea,which is consistent with the taxonomy that indicates the conservation of Dmrt4.In adult spotted scat,Dmrt4 is highly expressed in the testis,ovary and gill.In gonads,Dmrt4 was expressed significantly higher in phaseⅢandⅣtestes than those of phase V testis and phaseⅡ,ⅢandⅣovaries.Dmrt4 is expressed during the embryo developmental stages with the highest level at the gastrula stage.