Effect Of ACSL3 Gene On Differentiation Of Preadipocytes Of Chinese Red Steppe Cattle And Its Genetic Polymorphism

Long-chain acyl-coa synthetase(ACSLs)is an essential enzyme in the biosynthetic pathway,which helps to activate fatty acids,especially those acids with the chain length of 12-20 carbons.ACSLs is a complete initial step for almost all metabolic pathways in mammals,such as complex lipid synthesis,protein modification,and fatty acid beta-oxidation.ACSL3 is a member of the ACSLs family and contains high levels of polyunsaturated fatty acid phospholipids,which are widely expressed in the lipid droplets of the endoplasmic reticulum(ER)and most tissues.ACSL3 regulates the activity of key transcription factors for adipocyte differentiation,helps maintain lipid balance,and participates in the absorption of fatty acids.At present,the research on the mechanism and genetic polymorphism of ACSL3 gene in the differentiation of bovine preadipocytes into adipocytes is still unclear.In order to explore the regulation of the ACCL3 gene on the metabolism of bovine lipids and the function of genetic polymorphism,ACSL3 gene was used as the research object,the role of ACSL3 gene in the differentiation of precursor adipocytes into adipocytes was analyzed by constructing overexpression vector and siRNA method.Meanwhile,at the level of Chinese Red Steppe cattle population,the polymorphism of ACSL3 gene was detected by Sanger direct sequencing technology to analyze the correlation between its SNPs and the meat quality traits of beef cattle.The main research results are as follows:(1)The ACSL3 gene of Chinese Red Steppe cattle was successfully cloned,and related bioinformatics analysis was carried out.The mRNA and protein expression differences of ACSL3 gene in various tissues were analyzed.(2)The fat cells of Chinese Red Steppe cattle preadipocytes were successfully isolated,and the expression of mRNA and protein of ACSL3 gene in bovine preadipocytes was determined.(3)The overexpression vector and siRNA were constructed and transfected into bovine preadipocytes.Overexpression of ACSL3 gene significantly increased the mRNA and protein content of PPAR ? and C/EBP ? during adipogenic differentiation(P<0.05),while interference with ACSL3 gene significantly decreased the mRNA and protein content of PPAR? and C/EBP?(P< 0.05);ACSL3 gene overexpression can significantly increase the content of triglycerides and lipid droplets in the Chinese Red Steppe cattle fat cells(P < 0.05),while interference with ACSL3 gene can significantly reduce the content of triglycerides and lipid droplets in the cells(P < 0.05).(4)Taking the Chinese Red Steppe cattle as the research object,the SNP locus was detected in 15 exons of ACSL3 gene,and it was found that only the exon 6 had a G-A mutation at g.111870595(E6-G>A).Correlation analysis was carried out in combination with meat quality traits.It was found that there was a significant difference between the mutation and the peanut acid,linoleic acid,shear strength,fat content and water content of Chinese Red Steppe cattle.In summary,during the adipogenic differentiation of Chinese Red Steppe cattle,ACSL3 gene can promote the synthesis of triglyceride and lipid droplets in cells,promote the expression of adipogenic differentiation marker genes,and positively regulate adipocyte differentiation;Meanwhile,the mutation of ACSL3 gene g.111870595 G>A(E6-G>A)occurred in the coding region,which was correlated with fatty acid contents and some meat quality traits.The results of this study can provide basic materials for further exploration of the function and application of ACSL3 gene,and provide theoretical basis for relevant studies on bovine fat metabolism and fat deposition.