Reasearch On The Mechanism Of Mstn Gene In Reducing Fat Deposition In Fish

The fat content in the muscle has an important effect on the quality of farmed animals' meat.Increasing the fat content in the muscle to improve the tenderness and flavor of meat and so on is one of the hot research topicsin the breeding industry.Myostatin(mstn)is a member of transforming growth factor ?(TGF?)superfamily and also a negative regulator of skeletal muscle growth.Other researches have revealed that mstn may affect lipid metabolism and glycometabolism.So in this study we want to investigate the effects of mstn on intramuscular fat formation.We built the knock out model in loach and constructed a cellular model of intramyocellular lipid deposition in fat head millow muscle cell,we studied the role and mechanism of mstn in muscle fat synthesis and decomposition from two aspects between in vivo and in vitro experiments,through RT-PCR,Western blot,Oil Red O staining,lipidomics,and other biochemical and molecular detection methods.The pivotal results as follows:1.mstn gene deletion can significantly promote the development of muscle tissue,but at the same time,it can reduce the fat synthesis in muscle tissue.LC-MS analysis showed that the lack of mstn significantly affected the lipid componentsin loach muscle tissue especially decreased the triglyceride contents.Besides,muscle ATP contents of WT and MU were measured here,and we found that the ATP content in MU muscles was very significantly lower than that in WT muscles.Therefore,the way that mstn regulates fat metabolism may be through energy metabolism pathways.Next,we examined the expression of mitochondrial respiratory chain complex-related genesin muscle,no significant differences were found in the expression of marker genes ndufb8,ndufs1,and mtnd5,which represent the mitochondrial function,but the expression of complex II related genes in MU was increased significantly compared with WT,it showed that the lack of mstn does not affect the function of mitochondria and the electron transfer processin the process of mitochondrial respiration.However,the complexes III,IV,and V that involved in energy synthesis,the gene expression in the MU was significantly lower than WT,which is consistent with the results of ATP content detected.2.Based on the fat head millow(FHM)cell line and transfected by recombinant plasmid and si RNA,the knockdown cellular model of mstn in fish cells was successfully constructed.On the basis of the model,the fat deposition under mstn mutation was studied by adding different fatty acid to the medium.In a certain concentration range,the deposition of fat in myocytes increased within the increase of fatty acid exposure concentration.In addition to arachidonic acid C20:0,the other NC cells added with the some fatty acids showed significantly higher fat content compared with mstn knockdown group.At the same time,due to the different level of mstn gene expression,there are significant differencesin cell proliferation rate and cell morphology in the model construction,the specific performance is knockdown group > NC group.These indicators showed that the lack of mstn can promote the proliferation and development of fish cells,form a muscular phenotype,and also reduce intramuscular fat content.3.RT-PCR analysis showed the reduced expressions of fatty acid synthesis-related enzymes scd1,ppar?,and srebp1 c in mstn-defincient model(loach and FHM cell lines).In addition,after adding saturated fatty acids C16:0,C18:0 a nd oleic acid C18:1,compared with NC control group,the expression of fatty acid synthase(fasn,acc),key enzyme of triglyceride synthesis(dgat)and a ser ies of related enzymes of de novo fatty acid synthesis were inhibited in FHM cells after adding saturated fatty acid C16:0,C18:0 and oleic acid C18:1,and t he expression level of mstn was compensated after adding saturated fatty acid C20:0.These results also indicate that mstn plays an important role in the reg ulation of fatty acid synthesis pathway in FHM cells.Furthermore it also indic ated that mstn can reduce fat deposition by inhibiting fatty acid synthesis.How ever,the role of mstn in fatty acid synthesis,transport and other related pathw ays has not been known.In conclusion,this study investigated the role of mstn in muscle growth and fat metabolism form in vivo and in vitro experiments by using economic fish loach and fat head millow muscle cells as an experimental model of mstn mutation,and then explored the role in organismic mechanism briefly.The research methods and results have certain reference significance for further study of the function and metabolic mechanism in muscle,and it also provided an experimental basis for the development of the combination of molecular technology and genetic breeding.