Polymorphism Analysis Of ACTC1 Gene In Qinchuan Cattle And Its Effect On The Proliferation And Differentiation Of Myoblasts And Preadipocytes

In recent years,with the improvement of people’s living standards,high-quality beef is more and more favored by consumers.At present,how to improve the output and quality of beef has become an urgent problem to be solved.The growth and development of muscle tissue is directly related to the yield of beef,and the content of intramuscular fat(IMF)is an important decisive factor to improve the quality of beef.At the cellular level,the proliferation and differentiation of myoblasts and preadipocytes directly affect the growth and development of muscle and fat,and then affect the yield of beef.And the interaction between myocytes and adipocytes affects the formation of marbled beef and ultimately affects the quality of beef.Actin alpha cardiac muscle 1(ACTC1)is a differentially expressed gene screened by transcriptome sequencing technique in the co-culture of bovine myoblasts and preadipocytes in our laboratory.The effects of ACTC1 on bovine growth and meat quality traits,as well as on the proliferation and differentiation of bovine myoblasts and preadipocytes have not been reported.Therefore,in this study,the ACTC1 was used as the target gene to screen the SNP locus,and to analyze the relevance between the polymorphism of the gene and the growth and meat quality traits of Qinchuan cattle.In order to further analyze the effect of ACTC1 on the development of bovine muscle and adipose tissue,adenovirus-mediated interference,overexpression and ectopic expression were used to study the effect of ACTC1 on the proliferation and differentiation of bovine myoblasts and preadipocytes at the cellular level,so as to further reveal the mechanism of ACTC1 regulating the growth and development of beef muscle and adipose tissue.The experimental results and conclusions are as follows:1.The PCR-RFLP technique was used to classify the SNP site(30363938)of the fourth exon of ACTC1 gene,and then the typing results were correlated with the 12 body size traits of 339 Qinchuan cattle.The results showed that the polymorphism of this locus was significantly related to the rump length,ischial end width and ophthalmic muscle depth of the beef cattle(P<0.05).2.The results of ACTC1 expression characteristics showed that: ACTC1 was highly expressed in heart and muscle tissues of adult and newborn cattle,which was significantly different from that of other tissues(P < 0.05),but lower in adipose tissue;and the expression level in other tissues of adult cattle was significantly higher than that in newborn cattle(P <0.01,P < 0.05)except kidney and rumen(P >0.05).The expression trend of ACTC1 in the process of induced differentiation of myoblasts was increased first and then decreased;however,the expression of ACTC1 was not detected during the process of induced differentiation of preadipocytes.3.After interference and overexpression of ACTC1 in bovine myoblasts,the proliferation of myoblasts was significantly inhibited and the expression of P21 increased significantly,while there was no significant change in cell proliferation after overexpression of ACTC1 in bovine preadipocytes,and there was no significant change in the expression of P21,P27,CCNA2 and CCND2 proliferation marker genes in bovine preadipocytes.4.After interfering with ACTC1 in bovine myoblasts,myotube formation was significantly inhibited.The expression of myogenic marker genes was detected at the levels of m RNA and protein.It was found that MYOD1,MYOG,MYH3,MRF4 and CKM decreased significantly or very significantly during the middle and later stage of myotube formation(P < 0.05,P < 0.01),while MYF5 increased significantly or very significantly at D0,D4 and D8(P < 0.05,P < 0.01).MEF2 A decreased significantly on D0(P < 0.05)and D4(P < 0.01),but increased significantly on D2 and D6(P < 0.05).On the contrary,overexpression of ACTC1 significantly promoted myotube formation,especially on the maintenance of myotube in the later stage of cell differentiation.Ectopic expression of ACTC1 in bovine preadipocytes significantly promoted the production of lipid droplets,and lipogenesis related factors(FABP4,PPARγ,SCD1,FASN)were significantly up-regulated at m RNA and protein levels.In summary,this study found that the ACTC1 gene polymorphism is significantly related to some meat traits of Qinchuan cattle.ACTC1 can promote the differentiation of myoblasts and preadipocytes,and affect the proliferation of myoblasts.Based on this,it is suggested that the ACTC1 may be important candidate genes that affect intramuscular fat(IMF)production in beef cattle.