The Study Of METTL21C On Regulating The Mechanisms Of Skeletal Muscle Growth And Development In Chicken

Methyltransferase-like 21 is a distant member of the methyltransferase superfamily,including METTL21 A,METTL21B,METTL21 C and METTL21 D.METTL21C is expressed in all tissues of animal organism,and appears to be highly expressed in skeletal muscle specifically.The report revealed that METTL21 C can promote the myogenic differentiation of mouse C2C12 cells and inhibit muscle atrophy,but the function of METTL21 C in poultry skeletal muscle is relatively less studied.Therefore,this paper aimed to explore the affects mechanism of METTL21 C on the growth and development of skeletal muscle in poultry,to provide reference value for improving the quality of poultry muscle and production performance,and to lay a theoretical foundation for sports science,myopathy and metabolic homeostasis.In this study,primary chicken skeletal muscle satellite cells were isolated by enzymatic digestion from Lueyang black-bone chicken,and the effects of METTL21 C on myogenic differentiation were investigated in isolated primary cell model.METTL21 C interacting protein was identified by liquid chromatography-mass spectrometry and CoImmunoprecipitation.To further verify the role of METTL21 C binding with Hsc70 in myogenic differentiation.Finally,the study elaborates systematically the molecular mechanism of METTL21 C effecting on the growth and development in poultry skeletal muscle.The main results were as follows:1.Based on the analysis of transcriptome sequencing data of thigh muscle tissue from Lueyang black-bone chicken and literature consultion,we have found METTL21 C gene may affect the growth and development of poultry skeletal muscle.Further detected expression pattern of METTL21 C gene showed that METTL21 C was expressed in all tissues from Lueyang black-bone chicken,and the highest expression in myocardium,skeletal muscle comes second.At the same time,the expression of METTL21 C in skeletal muscle increased by the increase of animal age.2.Chicken primary skeletal muscle satellite cells were isolated by enzymatic digestion.The isolated primary skeletal muscle satellite cells were finally identified by detecting the expression levels of Pax7 and MyoG marker genes,which provided a cell model for subsequent experiments.3.METTL21 C overexpression vector was successfully constructed and transfected into chicken skeletal muscle satellite cells.The results showed that the expression level of genes involved in muscle development increased,indicating that METTL21 C could promote the growth and development of poultry skeletal muscle.4.The interaction proteins of METTL21 C were identified by LC-MS/MS.Then Hsc70 was selected as the research object.And the protein interaction between METTL21 C and Hsc70 by Co-IP technology.To further explore the effect of interaction between METTL21 C and Hsc70 on muscle development.We found that the expression of genes related to muscle development reduced after overexpression of Hsc70,suggesting it has the ability to inhibit myogenic differentiation,but the binding of METTL21 C and Hsc70 weakens the inhibitory effect of Hsc70 on myogenic differentiation.5.The K561 mutation vector of Hsc70 was constructed and the interaction between METTL21 C and Hsc70 was verified by Co-IP technique.The results reflected that METTL21 C did not bind to Hsc70 after mutation of K561 locus,indicating that K561 was the binding site between METTL21 C and Hsc70,which indicated that METTL21 C was involved in regulating the growth and development of poultry skeletal muscle by binding to K561 locus of Hsc70.