| MRF4,Myf5,Myogenin and MyoD are members of myogenic regulatory factors(MRFs)sharing a similar bHLH domain.Proteins of MRFs can homodimerize themselves or hetrodimerize with E-proteins and then bind to E-box(CANNTG)at the promoter region following dimerization and activate transcriptions of many skeletal muscle-specific genes.As transcriptional factors,MRFs play important roles in the processs of muscle development.Study towards them can help us better understand the key links that determine the formation of muscle.In order to gain insight into MRFs in mandarin fish,the cDNAs of MRF4 and Myf5 were cloned.Expression patterns of MRFs in various tissues,different embryonic stages and development stages of post-embryonic were analyzed.Finally,the effects of short-period fasting and reefeding on the expressions of four MRFs were studied.The main results were shown as follows:1.The complete sequence of MRF4 and Myf5 were cloned from mandarin fish by means of RACE method.The protein sequences were rather conserved in bHLH domain which was consisted of about 52 animo acids through alignments of the MRF4 and Myf5 genes.Phylogenetic tree analysis showed that the MRF4 and Myf5 in mandarin fish were associated with the same(Perciformes,Serranidae)into a grouper of orange-spotted grouper.It shows that mandarin fish had the closest genetic relationship with orange-spotted grouper.2.RT-PCR was used to study the different tissues distribution of four MRFs in mandarin fish.Results indicated that the four MRFs were mainly distributed in red and white muscle,the expression of the myogenic regulatory factors in white muscle was significantly higher than that in other tissues.In addition to expression of muscle,the were also observed in all tested sample including heart,brain,liver,spleen and kidney in mandarin fish.3.Quantitative RT-PCR was used to study the expressions of four MRFs in different embryonic stages and post-embryonic stages.Results indicated that the four MRFs were highly expressed from the blastular stage.Of the four MRFs,the expression of the MRF4 increased from the gastrular stage to muscular effect stage(P<0.05),Myf5 had began to descend after somite formation stage and the highest expression values of muscular effect stage.The expression of Myogenin and MyoD were significantly up-regulated in tail buds stage,muscular effect stage and hatching stage(P<0.05).4.The influence of four MRFs on short-period fasting and reefeding in mandarin fish by RT-qPCR method.The experimental results show,(1)in the 14d of short-period fasting and reefeding,the expression of gene MRF4 continually increase,and increase significantly then reach a peak on 7d(P<0.05),at last recovered to initial level on 14d.But the expression of genes Myf5,Myogenin and MyoD showed significantly increase(P<0.05)on the first day and last to 3d,and recovered to initial level on 5d,on the period of 7d to 14d showed an increasing tendency(P<0.05);(2)The results of short-period fasting and fed a single meal experimental show:the expression of genes MRF4,Myogenin and MyoD after satiation of mandarin fish showed up-regulation while Myf5 showed a diminishing trend in the beginning of 3h.The expression of MRF4 maintained stable in 3h to 24h,start increasing at 48h and recovered to initial level at 96h;Myf5 showed significantly increase at 6h(P<0.05),after that start decline,then maintained stable at 48h,at 96h showed significantly decline(P<0.05);Myogenin showed diminishing trend in the beginning of 12h,then increased to 48h,at last recovered to initial level at 96h;MyoD reached the highest expression at 1h,then continually decline to 12h,reached another peak at 24h,then showed diminishing trend(P<0.05).Those results are meaningful for researching the mechanism of muscle regulation of mandarin fish. |
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