Molecular Cloning And Analysis Of Myostatin Passthway Related Genes In Half-smooth Tongue Sole (Cynoglossus Semilavevis)

Half-smooth tongue sole(Cynoglossus semilaevis)is an important commercial fish, and has many strong points such as fast-growing in aquaculture. Myostatin, also known as GDF 8, is a member of the transforming growth factor-β(TGF-β) family and a strong negative regulator of muscle growth,mainly expressed in the embryo and adult skeletal muscle of mammals and other species. Follistatin(FST), which regulates TGF-βfamily members including Myostatin, is a cysteine-rich single chain peptide and an important inhibitor of MSTN; Activin II type B receptor(ActRIIB)is a member of the transmembrane serine / threonine kinase receptor family, Myostatin initially forms complexes with ActRIIB to activate downstream Smad transduction protein, thus starts signaling pathways. In this study, homological clone and RACE (Rapid amplification of cDNA ends) techniques were used to obtain full-length of Follistatin and ActRIIB cDNA, which were cloned and analyzed by bioinformatic softwares. In addition, the detailed investigations were carried out by Quantitative real-time PCR regarding the expression patterns of these two genes in different tissues and developmental stages respectively.In this study, the Follistatin gene was isolated from one of flatfish species, C. semilaevis. The full length cDNA sequence was 1373bp which contained a 16 bp 5' UTR, a 417bp 3'UTR, and a 966bp putative ORF which encodes a polypeptide of 321 amino acids with an estimated molecular mass of 35.5kDa. Sequence analysis revealed that Follistatin protein was composed of N-terminal domain, C-terminal domain, Follistatin domain I, Follistatin domainⅡ, Follistatin domainⅢand a potential signal peptide contained 31 amino acids. The deduced amino acid sequence showed high identity with other orthologs, meawhile Follistatin domainⅠand Follistatin domainⅡ, which contained cysteine-rich residues, were more conservative. Quantitative RT-PCR analysis indicated the mRNA expression of Follistatin could be detected in all 10 tissues, highly in spleen and gonads. The expression of Follistatin exhibited the similar?tendency with that of Myostatin in the developmental stage, indicating a certain interaction of the two genes during embryo development, which suggested that Follistatin may paly a role in the early development of half-smooth tongue sole.ActRIIB is an important binding receptor in the signal pathway of MSTN. Using the techniques of homological clone and RACE, the full-length cDNA was obtained, which contained a 195 bp 5'UTR, a 1539 bp open reading frame(ORF)encoding 512 amino acids and a 325 bp 3'UTR. The putative amino acid sequences with an estimated molecular mass of 35.5kDa had a potential signal peptide cotained 24 amino acid in N-terminal sequence. Sequence analysis revealed that there were three important domains: the extracellular domain, transmembrane domain and serine/threonine kinase domain. Also there were two distinct serine/threonine kinase active site signature sequences. The mRNA expression of ActRIIB could be detected in all 10 tissues in varying degrees. In the developmental stage, ActRIIB mRNA expression was maternal, showed gentle fluctuation and in a low-level compared with that of csMSTN.