CDNA Cloning Of Paramisgurnus Dabryanus MSTN And MyoD And Their Expression In Response To Dexamethasone Stress

Muscle growth and differentiation related genes mainly include the myostatin(MSTN)gene and myogenic regulatory factor(MRFs)family gene.MSTN gene,which belongs to the transforming growth factor β(TGF-β)superfamily,is a negative regulation gene of muscle development by inhibiting excessive proliferation of myoblasts.MRFs mainly include Myf5,MyoD,MyoG and MRF4,and the MyoD gene is one of the leading regulatory genes of vertebrate muscle development by regulating skeletal muscle formation and differentiation.MyoD gene deletion will block myoblast proliferation and differentiation.Fish stress will affect muscle growth.in order to understand the mechanism of stress affects the growth of fish,In this paper,paramisgurnus dabryanus as the research object cloned the cDNA sequence of MSTN gene and MyoD,respectively,subsequently analysis two sequences using some online softwares and local software.Tissue expression pattern of normal Paramisgurnus dabryanus MyoD gene and MSTN gene mRNA were detected and muscle tissue expression pattern of Paramisgurnus dabryanuswith continuous injectionof dexamethasone were detected at different times and dose of dexamethasone by real-time fluorescence quantitative PCR;The main results are as follows:(1)The length of the cDNA sequence of Paramisgurnus dabryanus MSTN gene is 1229 bp(GeneBank accession number:KY676785),which encodes a 375-amino-acids protein,its molecular weight is 42072.23da and theoretical isoelectric point pl=6.82.There are 9 conserved cysteine residues,an enzyme hydrolysis site(RIRR)and 11 phosphorylation sites in MSTN amino acid sequence.The prediction of secondary structure shows MSTN containsa signal peptide,a TGF-βpropeptide domain and a TGF-β mature peptide domain.MSTN protein sequence has the highest homology with rare Minow,reaching 96%.The length of the cDNA sequence of Paramisgurnus dabryanus MyoD gene is 951 bp(GeneBank accession number:KY676786),it encodes a 274-amino-acids protein,whose molecular weight is 30549.89da and whose theoretical isoelectric point pI=5.37.The prediction of secondary structure shows MyoD amino acid sequencecontains 26 phosphorylation sites and a basic helix loop helix(bHLH)domain which is a protein structural motif in MRF family.MyoD protein sequence has the highest homology with Culter alburnus,reaching 92.7%.(2)The expression pattern of MSTAN and MyoD genes mRNA areubiquitous but the expression was significantly different.MSTN gene mRNA is particularly highlyexpressed inthe muscle and particularly lowly expressed in the liver,spleen,gills,and heart,relatively.MyoD gene mRNA is observed at the highest levels in muscle and lowest levels in the heart,liver and spleen,relatively.(3)After five days of continuous injection of different concentrations of dexamethasone on the Paramisgurnus dabryanus,the mRNA expression of MSTN gene and MyoD gene in the experimental group(60 g/kg,600 g/kg,1200 g/kg)were significantly lower than that of the control group(0 g/kg)(P<0.05).The expression of MSTN gene in experimental group mRNA increased with the increase of the concentration of dexamethasone but the difference between 600 g/kg and 1200 g/kg group was not marked(P>0.05).However,the expression of MyoD gene mRNA in the experimental group decreased markedly with the increase of dexamethasone concentration(P<0.05).The results showed that the expression of MSTN and MyoD genes was affected by the concentration of dexamethasone in a dose dependent manner.(4)After continuous injection of 600 g/kg dexamethasone,the mRNA expression of MSTN gene in the experimental group was significantly decreased at 6h,12h,5d and 10d(P<0.05),while the mRNA expression of MSTN gene in the experimental group was significantly higher than that in the control group(P<0.05).The mRNA expression of MyoD gene was decreased at the 6th,12th and 24h(P<0.05),but the expression of MyoD mRNA was decreased at the 5th day and 10th day,but the difference was not significant(P>0.05).The results showed that the expression of MSTN and MyoD gene was time-effect after dexamethasone stress treatment.Short-term stress could inhibit the expression of MSTN gene and promote the expression of MyoD gene;The long-term stress induced the expression of MSTN gene and MyoD gene were inhibited.