Cloning And Expression Of Muscle Growth Factor In The Seriola Aureovittata

In this paper,Seriola aureovittata was used to clone the muscle-specific genes by RACE,including myogenic regulatory factors(MRFs)and myosin heavy chains(MyHC),myostatin(MSTN),phosphatase and tensin homolog(PTEN)cDNA sequences.Their gene structure,amino acid sequence and tissues expression is analyzed and their temporal spatial expression patterns during embryonic stages and juveniles was investigated by quantitative real-time PCR(qPCR).The role of these genes in temperature and different age stages was discussed by qPCR,hormone radioimmunoassay for muscle-specific genes and growth hormone(GH),insulin growth factors I and II(Insulin-Like growth factor I,II;IGFI,II).This study provides a theoretical basis for the early growth and development of Seriola aureovittata and good seed culture techniques.The main findings are as follows:1.Cloning and tissue expression analysis of muscle-specific genes in Seriola aureovittataThe full-length sequence of the muscle-specific gene of the Seriola aureovittata was cloned from the muscle using RACE methods.The MyoD1 cDNA was 1233 bp in length,and the open reading frame was 897 bp,encoding 298 amino acids;the MyoD2 cDNA 900 bp in length,the open reading frame was 792 bp,encoding 263 amino acids;the MRF4 cDNA was 951 bp in length,and the open reading frame was 720 bp encoding 239 amino acids;Myf5 cDNA is 951 bp in length,and the open reading frame is 801 bp,encoding 266 amino acids;MyoG cDNA is 1105 bp in length,and open reading frame is 753 bp,encoding 250 amino acids.And all of the above MRFs family members contain two conserved domains,one is the HLH domain and the other is the extension region Basic region.Homology analysis found that MRFs have the closest relationship with Seriola dumerili,and they are clustered with Perciformes and Pleuronectiformes.,which is far from mammalian.The MyHC cDNA of the Seriola aureovittata was 6143 bp in length,and the open reading frame was 5811 bp,encoding 1936 amino acids,and the myosin heavy chain had 4 ATP binding sites according to its structure.These amino acid residues constitute two conserved domains of MyHC,the head region MYSc-Class II and the tail region Myosin tail1 domain,while the Nterminus contains the domain Src homology region 3(Srchomolo-gy3,SH3).The homology analysis showed that the homology of MyHC and Seriola dumerili was as high as 98.0%,and it was highly conserved with the Perciformes.The MSTN cDNA of Seriola aureovittata was amplified 1828 bp in length,and the open reading frame was 1131 bp,encoding 376 amino acids.MSTN contains two conserved regions: TGF-? propeptide region and TGF-? domain,a total of 13 cysteine residues,a glycosylation site in the sequence,located in the 73 rd amino acid,the first 22 Amino acid residues are signal peptide sequences.The PTEN cDNA was 1575 bp in length and the open reading frame was 1287 bp,encoding 428 amino acids.The PTEN amino acid sequence comprises two major domains: the PTP(PTPc)catalytic motif and the PTEN-C2 domain.The PTEN sequence contains eight cysteine residues and does not contain a signal peptide and a glycosylation site.The MRFs family of the Seriola aureovittata were most expressed in muscle tissue,and were significantly different from other tissues(P<0.05),and were slightly expressed in brain,pituitary,liver and other tissues.MyHC has a low expression level in brain,pituitary,liver and other tissues,and the highest expression in muscle tissue,and is significantly different from other tissues(P<0.05).MSTN mRNA expression was highest in muscle(P<0.05),followed by higher expression in kidney,and lower expression in brain,pituitary,kidney,head kidney,stomach,intestine and other tissues.PTEN mRNA was expressed in the tissues examined,with the highest expression in the heart(P<0.05);higher expression was also detected in the spleen,head kidney,kidney,stomach,and intestine;in the liver,muscle,brain,pituitary,the expression was not significant(P<0.05).The high expression of muscle-specific genes in muscle suggests that it plays an important role in the skeletal muscle of the Seriola aureovittata.2.Differential expression of muscle-specific genes in the early growth stage of the Seriola aureovittataThe expression of MRFs was detected in all stages of embryonic development.The expression levels of MyoD1 and MyoD2 mRNA were significantly increased in the neural stage.MRF4 mRNA showed irregularity before the embryo encompassing yolk 50%.The fluctuation trend showed a sudden and significant increase in the expression level of encompassing yolk 70%(P<0.05),and then decreased significantly to the lower expression level.The expression of Myf5 from the early gastrula significantly increased,and reached the peak value in encompassing yolk 50%(P<0.05);MyoG had the highest expression level in the encompassing yolk 70%(P<0.05),and then decreased significantly.The expression level of MyHC was higher before the 16-cell stage,and then decreased significantly.The expression was suddenly increased in the encompassing yolk 70%,and the expression level was the highest in the hatching stage(P<0.05).The expression of MSTN increased significantly from the encompassing yolk 70% to the hatching stage,and reached the peak in the hatching stage(P<0.05).The expression level of PTEN increased gradually from the ow blastula stage to the peak in the encompassing yolk 70%(P<0.05),and maintained a high expression trend.During the development of the juveniles,the expression of MyoD1 was significantly increased at 20d(P<0.05),then decreased gradually,and remained high at 30 d and 40 d.The expression level of MyoD2 was higher before 15d(P<0.05).The expression level of MyoD1 was higher than that of MyoD2 except 2d and 6d.The expression level of MRF4 was lower at 0-40 d and increased significantly at 45d(P<0.05).Myf5 increased significantly at 20d(P<0.05),and then increased to a peak at 30 days.The expression level of MyoG increased gradually after hatching,and peaked at 25d(P<0.05).The expression of MyHC increased significantly at 20d(P<0.05)and maintained a high expression trend to 40 d.The expression level of MSTN increased at 25 d to 30d(P<0.05).PTEN suddenly increased significantly(P<0.05)at 20 d,then gradually decreased and remained lower to 60 d.The above results indicate that muscle-specific genes may play an important role in the regulation of early growth and development of the Seriola aureovittata.3.Regulation of muscle-specific genes and growth axis(GH/IGF axis)on Seriola aureovittata of different specifications and agesThe results of PCR showed that MyoD1,MyoD2,Myf5 and MyoG of the MRFs family had the highest expression at 400 g,followed by a downward trend.MRF4 showed an increasing trend and the highest expression at 500 g.MyHC has the highest expression level during the 500 g.The expression of MSTN and PTEN mRNA in muscle increased with the size of weight,and the highest in 500 g.The results of radioimmunoassay and PCR showed that the expression of GH mRNA in pituitary gland and plasma GH had the same trend,and the highest expression level in 400g;the expression level of IGFI mRNA in liver was consistent with the trend of plasma IGFI.The expression trend reached a peak at 400 g and decline.The expression level of GH in muscle decreased and then increased.The expression level of IGFI in muscle increased and then decreased.At 400 g,the expression level of IGFII in muscle decreased and then increased.At different ages,MRFs and MyHC were the highest in the 2 year,MSTN was the lowest in the 2 year,and PTEN was the highest in the 2 year.The results of plasma hormone radioimmunoassay and PCR showed that the expression level of GH in the pituitary was similar to that of plasma GH,and the expression level of GH was the highest in 2year;the expression level of IGFI in the liver was consistent with that of plasma IGFI,which was the peak of the expression in the 2 year.The expression levels of GH,IGFI and IGFII in muscle increased and then decreased with age,and were highest in 2 year.These results indicate that muscle-specific genes and growth factors jointly regulate the growth of Seriola aureovittata,and these factors can participate in the regulation of the growth through various pathways such as autocrine,endocrine and paracrine.