Research Of Muscle Development Of Blunt Snout Bream And Evolutionary Analyses Of MRFs Family

Megalobrama amblycephala is one of important herbivore economic fish in our country.Now due to overfishing,its wild population drastically reduced,and germplasm resources were degradated,so it is important to strengthen the breeding of good varieties.Characteristics of the muscle fibers are closely related with the meat quality of animals,and the development of fish muscle fibers is different from mammals,which fish muscle fibers are recruited throughout the entire life cycle,however muscle fiber numbers have been fixed before the birth in mammals.Therefore,interpretation of the change rule of fish muscle fiber development and its molecular mechanism will be very important in revealing the genetic regularity of fish economic characters and the breeding of good varieties.In our study,the characterization of muscle fiber development and the proliferation and differentiation of muscle satellite cells in M.amblycephala was described by histology and immunology methods.Myogenic regulatory factors(MRFs)related to the growth of muscle fibers in M.amblycephala were cloned and characterized.The origin and evolution of MRFs and its function in the growth of muscle fibers were preliminarily analyzed.Moreover,the effect on muscle growth of M.amblycephala by different factors(Heat shock protein,HSP70;Lipopolysaccharide,LPS)was investigated.There results will be helpful for understanding the molecular mechanism of growth and development in M.amblycephala.The main results were as follows:1.The results about muscle fiber development in M.amblycephala indicated that with muscle development,fish body length,body weight,muscle fiber length,the diameters and numbers of red and white muscle fibers,and the areas including hyperplasia and hypertrophy area significantly increased.Moreover,red muscle fibers were initially located near the horizontal septum and appeared monolayers after hatching in M.amblycephala,later stratified hyperplasia and mosaic hyperplasia appeared at 15 dph(days post-hatching,dph).Subsequently,they gradually became multilayers and formed a red muscle group.Mosaic hyperplasia significantly strengthened at 20 dph in those fibers with increasing of smaller muscle fibers.Furthermore,body height was significantly increased and the fish shape became much closer to adult at 35 dph.In addition,we found that compared with 1-year-old M.amblycephala,the diameter scope of epaxial,horizontal septum and hypaxial muscle fibers including red and white muscle in 2-year-old fish exhibited broader,with dramatic reduction in frequency distribution of smaller diameter(<20 μm)and observable increase in frequency distribution of larger diameter(>50 μm).Additionally,the results based on immunofluorescence showed that more satellite cells existed in red muscle than white muscle in 1-year-old fish,rather than 2-year-old fish.The transmission electron microscope(TEM)results indicated that,muscle fibers could be divided into two kinds by morphological structure,one located in the outer edge strip of muscle fibers(Ribbon-like myofibrils,RM),the other located in the centre of muscle fiber(Polygonal myofibrils,PM).During muscle growth,the long axis of RM gradually increased,nevertheless no predominant change was detected in the minor axis of RM,the long and minor axis of PM.Moreover,the long and minor axis of nuclear also showed no significant difference.2.The cDNA sequences of MyoD,Myf5,MyoG and MRF4 of M.amblycephala were obtained by reverse transcription PCR(RT-PCR)and rapid amplification of DNA ends(RACE).MyoD and Myf5 were 955 bp and 723 bp,containing completeopen reading frame(ORF)of 828 bp and 723 bp which encoded proteins of 275 aa and 240 aa,respectively.Moreover,MyoG consisted of a 1,367 bp sequence which contained 24 bp of 5’ untranslated region(5’-UTR),a 765 bp ORF encoding a polypeptide of 254 amino acids and a 578 bp 3’-UTR including a polyA signal sequence.MRF4 was 1196 bp,containing a complete ORF of 720 bp which encoded protein of 239 aa.By comparing amino acids of various genes,bHLH、His/Cys-richand Helix Ⅲ domains,we deduced that the highly homology with M.amblycephala MRFs was other fishes,thereinto M.amblycephala MRFs had the highest identity with the homologues of Ctenopharyngodon idella and Cyprinus carpio and the lowest identity with the homologues of Homo sapiens and Mus musculus.MyoD and Myf5 were predominantly expressed in white muscle,followed by heart and brain,lowest in gonad and kidney.High expression of MyoD and Myf5 was detected in inclinator dorsalis muscle,caudal muscle and ventral muscle.MyoG was only expressed in gonad,white muscle,spleen,brain and liver,with the highest level in white muscle.Similarly,MRF4 mRNA was highly detected in white muscle,and almost undetectable in kidney and the brain.Moreover,the mRNA level of MRF4 was highest in eye muscle among muscle from different position.MyoD and MRF4 shared similar trend in 11 different stages of development.Both were detected highest at 10 dph and 20 dph,and the mRNA levels of MyoD also peaked at early gastrula,gradually decreased to hatching,later increased to 10 dph.MyoG mRNA was first detected at the segmentation period which was later than other three MRFs.The expression of MyoG mRNA in the segmentation period and at 12 hph was significantly higher than at other development stages.Furthermore,the expression of Myf5 mRNA in the segmentation period was also significantly higher than at other development stages.3.The analysis results of origin and evolution of MRFs of members showed that all of MRFs(322 bilateria MRFs)obviously fall into five categories,it was indicated that the homologous amino acid sequences of invertebrate MRFs were out-group of the vertebrate MRFs.We found that the origin of Myf5 and MyoD clusters was Eptatretus burgeri MyoD.Another interesting thing was that MyoG and MRF4 clusters were originated from Latimeria chalumnae MRF4-1,a "living fossil",nevertheless MRF4-2 was gathered closely with other vertebrate MRF4.From the genome of Bilateral animal,we found four members of MRFs,thereinto only three MRFs(MyoD,MyoG and MRF4)existed in Agnatha.However,also only three MRFs(MyoD,Myf5 and MyoG)were found in invertebrates.Interestingly,two kinds of MRFs homologous genes(Sp-MyoD,Sp-MyoD2 and Amphi-MyoD,Amphi-MyoDl)were found in Strongylocentrotus purpuratus and Branchiostoma floridae,respectively using bidirectional matching method in the genome data of S.purpuratus and B.floridae.The linear analysis result of MRFs indicated that four members of MRFs belonged to orthologous genes in the selected species.Moreover,the analysis results of origin and evolution of MRFs domains showed that TAD and H/C domains firstly appeared in Cyclostomata(E.burgeri)and Annelida(Capitella teleta),respectively-However,the conserved domains of bHLH and Helix III firstly appeared in Aschelminthes(Caenorhabditis elegans).4.In our study,the prokaryotic expression vector of pET28a-HSP70 was successfully constructured and active HSP70 recombinant protein was obtained.The mRNA levels of related genes had dramatically changed at one day after different concentrations of HSP70-injection in M.amblycephala.Thereinto,the expression of MyoG gene significantly increased in high concentration group(3.4 mg/mL),nevertheless observably decreased in the mRNA levels of MyoD,Myf5 and MRF4.With the increasing of HSP70-injection concentration,MSTNa and MSTNb mRNA firstly decreased and then increased,but MSTNb mRNA was dramatically higher than MSTNa.Furthermore,the frequency of satellite cells in low concentration group(1 mg/mL)was observed predominant higher than other concentration groups in both of red and white muscle.The result of gene mRNA expression in M.amblycephala skeletal muscle after LPS-injection showed that the expression of MyoD、Myf5 and Pax-7 gene were significantly higher in high concentration group(1.2 mg/mL)and control group at 7 d after LPS-injection in red muscle.Moreover,with the increasing of LPS concentration,the mRNA levels of MyoG and Pax-7 firstly increased and then decreased,nevertheless observably decreased in the mRNA levels of MyoD,Myf5 and MSTNb at 3 d after LPS-injection in white muscle.Furthermore,a dramatic increase for the mean frequency of satellite cells between the control group and LPS-injection groups in red muscle was found.However,the mean frequency of satellite cells was dramatically higher in low concentration group(0.6 mg/mL)at 3 d after LPS-injection,nevertheless no significant difference was observed in high concentration groups.