Molecular Characterization Of MstnⅡ Gene In Sparus Macrocephalus

MSTN is a functional glycoprotein which is secreted from histiocyte. Being a TGF-βsuperfamily member, MSTN plays an important role as a negative regulator in muscle growth. Natural mutation of MSTN gene in mice shows double-muscle phenotype and the weight of muscle tissue in gene knocked -out mice will increase by 2-3 times. Because of the importance of MSTN in animal breeding as well as scientists pay more attention to its potential application in animal breeding, we can improve the muscle content of cultured species, promote the growth of cultured species by inhibiting the activity of MSTN.Two types of MSTN have been recently found in some teleosts because of Gene duplication. Based on the strategy of homology cloning, MSTNⅡgene of Sparus macrocephalus ranging from start codon to stop codon which contains 3 Exons and 2 Introns was cloned from muscle tissue. In addition, using Sparus macrocephalus DNA as a template, we have amplified 562 bp 5'-UTR (5'-untranslated region) by Genome Walking Kit. Meantime, using Sparus macrocephalus brain tissue cDNA as a template, we have obtained 1080bp complete ORF (opening reading frame) and 160bp 3'-UTR (3'-untranslated region) of MSTNⅡby RT-PCR and RACE technique (Rapid Amplification cDNA Ends, RACE). It is the first report of Sparus macrocephalus MSTNⅡin China.The results of the sequence analysis show a big difference between MSTNⅠand MSTNⅡ, including the length and similarity of both sequences. However, ExonⅢis highly conserved in both length and similarity. It reflects that ExonⅢin MSTNⅡis highly conserved among species. The MSTNⅡshows a typical characteristic as a TGF-βsuperfamily member. The MSTNⅡcontains three domains (a potential signal sequence (residues 1-16), a TGF-βpropeptide domain (residues 33-241), and a TGF-β(262-359) domain), encodes 359 amino acid. There is a RXRR proteolytic processing site in C-terminal of TGF-βpropeptide domain and nine conserved cysteine residues in TGF-βdomain which are common in all TGF-βsuperfamily members.Detect MSTNⅠand MSTNⅡ′expression in 10 tissues of adult Sparus macrocephalus (muscle, brain, eyes, heart, liver, kidney, intestines, spleen, fat) via conventional RT-PCR with a pair of primers designed crossing intronⅠ.The results showed that MSTNⅠwas mainly expressed in muscle, brain, eyes and heart, while MSTNⅡwas mainly expressed in brain and adipose. Expression of MSTNⅡwas not detected in muscle tissue. The expression pattern suggested that MSTNⅠand MSTNⅡhave different function. MSTNⅠmay mainly inhibit the growth of muscle and MSTNⅡmay be related to the development of brain and adipose.