松江鲈MRF4基因克隆与组织表达分析

Cloning and Tissue Expression Analyses of MRF4 Gene in Trachidermus Fasciatus

MRF4(myf6或herculin)基因属于MyoD家族成员之一,为成肌细胞分化成肌管的重要调控因子.本实验以松江鲈(Trachidermus fasciatus)肌肉总RNA为模板,采用RT-PCR结合RACE的方法,获得1 049 bp全长cDNA序列,包含了1个含有720个核苷酸的开放性阅读框(ORF),共编码239个氨基酸.其编码区序列与石斑鱼同源性最高、为91.81%,其次是东方红鳍鲀、为83.40%,与哺乳类、鸟类的同源性在65%左右,与爪蟾同源性最低仅为59.65%.预测松江鲈MRF4蛋白具有保守的HLH结构域,bHLH结构域形成剪刀状α-螺旋二聚体.用荧光定量PCR方法检测发现,MRF4基因的mRNA在松江鲈6种组织中均有表达,但其在肌肉组织中表达量极显著高于其他组织,提示其在肌肉的生长发育过程中起着重要作用.

The transcription factor MRF4 is one member of MyoD family which have essential functions in myogenic lineage determination and muscle differentiation. In order to obtain full-length MRF4 gene sequence and characterize its tissue expression profile in Trachidermus fasciatus, RT-PCR and rapid-amplification eDNA ends （ RACE ） were used. A full-length of 1049 bp MRF4 cDNA,whieh contained a 720 bp long complete ORF encoding a 239 amino acid peptide. Multiple sequence comparison showed that the T. fasciatus MRF4 had a high homology with Epinephelus coioides at 91.81% ,followed by Takifugu rubripes. A homology with mammals and birds was at about 63%, and of the lowest homology with Xenopus laevis at 59.65%. The bHLH domain of T.fasciatus MRF4 was forming （x-helix dimer that like a scissors, and HLH sequence was highly homologous among different species. MRF4 gene was detected in all 6 examined tissues, but its expression level in muscle was significantly higher than the amount of other tissues, implying that the fish MRF4 gene would play an important role in muscle development.