Molecular cloning and expression analysis of myostatin gene in Carassius auratus in Qihe River

Myostatin (MSTN) is a negative regulatory factor in animal muscle development and growth. In order to identify the sequence information and the function of Carassius auratus MSTN in the process of muscle development, rapid amplification of cDNA ends (RACE) and real time PCR were used to clone the full length cDNA sequence and analyse the expression level of MSTN in embryonic development stages and different tissues. The results showed that the full length cDNA of MSTN was 2094 bp (no. KC851952.1), including 86bp at 5'-UTR, 880 bp at 3'-UTR, and a 1128 bp open reading frame encoded a peptide of 375 amino acids. The putative peptide contained a 22 amino acids signal peptide, a TGF-β propeptide domain (34-256), a TGF-β domain (281-375), a conserved RIRR proteolytic processing site and 9 conserved cysteine residues in the C-terminal of the protein. Multiple sequence comparison indicated that the MSTN of Carassius auratus has high similarity with cypriniformes species, while has low similarity with mammalian and bird. The phylogenetic analysis also showed that the crucian carp MSTN has close relationship with cypriniformes species like Carassius carassius, Cyprinus carpio and Labeo rohita. Real time PCR results showed that MSTN mRNA was checked in eight different tissues of crucian carp, among which the maximum level was detected in brain, followed by muscle and liver, the minimum level was displayed in intestine. In addition, the MSTN was also confirmed to express at every stage of embryogenesis, in which the highest level was found at fertilized eggs, followed by blastula and the lowest level was detected at neurula. Thus, the MSTN gene was speculated to have relation with crucian carp muscle development and growth, and might play an important role in the "double dorsal muscle" formation.