Cloning and expression analysis of the myostatin (MSTN) gene in Paralichthys olivaceus

Myostatin(MSTN)gene was isolated from Japanese flounder(Paralichthys olivaceus)by homology cloning strategy and genome walking.Two pairs of degenerate primers were designed according to the conserved region of MSTN genes from different fish.Full length DNA sequence of MSTN gene was obtained after one step of 3' genome walking and two steps of 5' genome walking.By sequence analysis and verified in cDNA level,Japanese flounder MSTN gene included three exons and two introns,encoding a 374-amino-acid protein.Eight TATA boxes,one CAAT box and six E boxes were located in 5' flanking region.A polyA signal existed in 3' flanking region.Two single nucleotide polymorphism(SNP)sites were discovered within MSTN cDNA,one located in coding region with synonymous mutation and the other located in 3' untranslated region.Nine conserved cysteine residues and an RXXR proteolytic cleavage domain were detected in the C terminal of protein.MSTN in Japanese flounder was highly homologous with MSTN genes in fish via phylogeny analysis,which had 64.5%,63.5%,64.5%,65.5%,80.1%,83.5%,84.5%,92.6%,90.5%,77.5% identity with human,house mouse,chicken,zebrafish-2,zebrafish-1,rainbow trout-2,rainbow trout-1,striped bass,red seabream and channel catfish,respectively.The MSTN gene of Japanese flounder might belong to type I MSTN,according to identity analysis.The RT-PCR analysis demonstrated that MSTN was undetectable during embryo development,indicating low expression or no expression during embryo development.MSTN had high expression in brain of Japanese flounder,indicating the important role in neural development.The expression of MSTN in kidney indicated the potential role in immunity.However,the expression of MSTN of Japanese flounder in muscle was low.Starvation could induce reduction of MSTN expression in muscle.Unspliced transcript was discovered in normal individual,which was verified by sequencing.The spliced transcript was about 1 270 bp and the unspliced transcript was about 2.4 kb which had all introns.The transcription of myostatin was influenced by environment factors in adult,indicating the transcription was under regulation.