Epigenetic Regulation Mechanism Of Cofilin Family CFL1 And CFL2 Gene Regulate The Development And Differentiation Of Bovine Myoblasts

According to the latest 2018-2025 Chinese Beef Industry Market Research Report,the consumption of beef is greatly increased in China,it reveals that potential demand and huge market space of beef market.Growth and development of muscle is the important factor in meat production and meat quality.Therefore,it is very important to research the molecular regulation mechanism of meat traits from the angle of genetics and epigenetics.Our project takes Qinchuan cattle as the research object,First,we analyzed the expression patterns of CFL1 and CFL2 genes in different tissues of Qinchuan cattle at different development stages,and determined the tissue expression distribution of CFL1 and CFL2 genes in Qinchuan cattle.;Then the DNA methylation levels of Qinchuan cattle at different developmental stages were determined by using hydrogen sulfite sequencing PCR(BSP)and heavy sulfite treatment and enzyme-cut analysis(COBRA)technology.and then revealed the binding site between micro RNAs and CFL1/CFL2 gene by real-time fluorescence quantitative PCR(QRT-PCR),dual-luciferase reporter system and Western blot(WB),and the effects of micro RNAs on cell differentiation are preliminarily studied.Interference and other techniques to clarify the regulation mechanism of CFL1 and CFL2 gene on the differentiation of muscle cells;Finally,a population genetic analysis was carried out to reveal the regulation mechanism of CFL1 and CFL2 gene on bovine muscular development.The main research contents and results are as follows:1.Spatio-Temporal expression patterns of CFL1 gene and CFL2 geneFirst,we studied the expression of CFL1 and CFL2 gene in different tissues(heart,liver,spleen,lung,kidney,fat and muscle)in different stages of Qinchuan cattle(fetal bovine,calf,adult cattle)by q RT-PCR.The results showed that the CFL1 and CFL2 genes were expressed in different tissues of the development of Qinchuan cattle,and the expression patterns differed from each other.And with the growth and development of muscle tissue,the expression level of CFL1 gene showed a significant downward trend,while the expression level of CFL2 gene showed a significant upward trend.2.Study on DNA methylation level of CFL1 gene and CFL2 geneIn order to verify the epigenetic modification of methylation level and the effect of CFL1 and CFL2 gene on muscle growth and development by using BSP and COBRA technology.We found that: CFL1 gene coexist 3 Cp G islands(Cp GIs),including the 2 Cp GIs in gene body region and 1 Cp GI in the promoter region,and the promoter Cp GI contains 13 CPG sites,and CFL1 gene promoter in adult bovine muscle tissue methylation level is significantly higher than fetal bovine period;CFL2 genes coexist 2 Cp GIs,including 1 Cp GI in gene body region and 1Cp GI in the promoter region,and the promoter region Cp GI contains 8 CPG sites,and the promoter of the CFL2 gene in adult bovine muscle tissue is significantly lower in the methylation level than fetal cattle.The results indicated that the methylation level of muscle at different growth stages may be one of the factors that affect the expression of CFL1 gene and CFL2 gene.3.Target regulation of CFL1 gene and CFL2 gene by non-coded RNATo further confirm the effect of epigenetic modification of CFL1 and CFL2 gene on the growth and development differentiation of myoblasts,we found miR-182 and miR-183 can directly target the regulation of CFL1 and CFL2 genes respectively by using qrt-pcr,WB and build a double fluorescent enzyme reporting system.and miR-182 and miR-183 were the same micro RNA family clusters,and we also found that miR-182 and miR-183 negative regulate the expression of CFL1 gene and CFL2 gene respectively.After transfected miRNAs mimics and inhibitors into bovine myoblasts,we induced cell differentiation for 4 days,it was found that miR-182 and miR-183 could inhibit the differentiation of bovine myoblasts cells.4.Effects of CFL1 and CFL2 gene on the differentiation of myoblastsTo verify whether the CFL1 and CFL2 genes could regulate the differentiation of bovine myoblasts,we constructed overexpression and knock-down vectors of CFL1 and CFL2 gene,then transfected into bovine myoblasts respectively.After inducing differentiation 1d,3d,5d,7d,we detected the expression of CFL1 and CFL2 genes as well as muscle differentiation-related factors MYOD,MYOG and MYH3 to identify the specific regulatory mechanisms of CFL1 and CFL2 genes in the differentiation of bovine myoblasts.It was found that in the process of differentiation of bovine myoblasts,the effect of overexpression or knock-down CFL1 gene was not significant,while CFL2 gene could significantly increase the expression of muscle differentiation-related factors myod,myog and MYH3,and knock-down CFL2 gene could obviously inhibit the correlation factor MYOD,MYOG and MYH3.It is indicated that CFL2 gene plays a catalytic role in the differentiation of bovine myoblasts.5.Genetic variation of CFL1 and CFL2 gene and its correlation with growth traits ofQinchuan cattleFinally,we analyzed the genetic polymorphism of CFL1 and CFL2 gene in population genetics level by sequencing 488 Qinchuan bovine DNA pools.and we found 4 coded mutations in the exon 2 of CFL1 gene,named T-2052-C,T-2084-G,G-2107-C and T-2169-C,respectively.At the same time,we found 2 coded mutations in exon 4 and 1 non-code mutation in intron 2 of CFL2 gene,named G-1500-A,T-1694-A and C-2213-G respectively.The results of correlation analysis showed that the 4 mutation sites of CFL1 gene in Qinchuan cattle were correlated with 4 growth traits-body length,chest width,chest depth and rump length.The 3mutation sites of the CFL2 gene are associated with 6 growth traits of Qinchuan cattle-rump length,chest girth,chest width,chest depth,hip width and body weight.