Effect Of ACSL1 Gene On Unsaturated Fatty Acid Synthesis In Bovine Adipocytes

With the rapid development of China’s beef cattle industry and the continuous improvement of people’s living standards,high-quality meat such as"snowflake beef"is favored by the majority of consumers.Intramuscular fat deposition and fatty acid composition of beef affect beef quality.A high proportion of unsaturated fatty acids is related to the flavor of beef,which is helpful to improve the nutritional value of meat.Long-chain acyl CoA synthetase 1（ACSL1）can catalyze the acylation of ATP-dependent fatty acids to acyl coenzyme A,which is the first step of fatty acid synthesis and entry into cells.Association analysis showed that the gene variation in intron 5 of ACSL1 gene was significantly correlated with the relative content of fatty acids in skeletal muscle.In addition,previous studies found that the core promoter of bovine ACSL1 gene specifically binds a number of transcription factors related to the synthesis of unsaturated fatty acids in adipocytes,and cooperatively regulates the expression of ACSL1 gene,which means that ACSL1 gene may play an important role in the synthesis of unsaturated fatty acids in bovine intramuscular adipocytes.However,the mechanism of ACSL1 gene regulating the synthesis of unsaturated fatty acids in bovine intramuscular preadipocytes has not been reported.In this study,Qinchuan cattle adipocytes were taken as the research object.Adenoviral overexpression and siRNA interference with ACSL1 gene were used to clarify the effect of ACSL1 gene on the synthesis of unsaturated fatty acids in bovine intramuscular preadipocytes according to the expression of downstream target genes and the changes of fatty acid composition in adipocytes.The specific test results and conclusions are as follows:1.The CDS sequence of ACSL1 gene with length 2100 bp was amplified,and a siRNA-ACSL1 sequence targeting ACSL1 gene with high interference efficiency of86%was obtained by screening.The adenovirus vector pAdeno-EF1A（S）-mNeonGreen-CMV-ACSL1-3FLAG containing ACSL1 gene was constructed.High titers of overexpressed adenovirus AD-ACSL1 and AD-NC（no-load virus）were obtained by packaging and large-scale amplification,whose titers were 2.37x10¹⁰PFU/mL and 2.08x10¹11 PFU/mL respectively.2.The temporal expression of ACSL1 gene and unsaturated fatty acid synthesis related genes FADS1,FABP3,FASN,PPARγ,and SCD1 during bovine adipocyte differentiation was analyzed,and the results showed the expression levels of ACSL1gene and unsaturated fatty acid synthesis related genes FADS1,PPARγ,SCD1 and FASN were the highest on the fourth day of bovine adipocyte differentiation,and showed a trend of rising at first and then decreasing,while FABP3 had no significant change during bovine adipocyte differentiation.3.Overexpression of ACSL1 gene in bovine adipocytes upregulated the expression of FABP3,PPARγ,SCD1,ACLY and FASN,and down-regulated the expression of CPT1A.Oil red O staining showed that lipid droplets in bovine adipocytes increased after overexpression of ACSL1 gene.4.Interfering with ACSL1 gene in bovine adipocytes,the expression of FABP3and PPARγwas down-regulated,while the expression of FADS1 and FASN was up-regulated,while the expression of SCD1 was increased at first and then decreased.The proportion of C18:0 in adipocytes decreased significantly（P<0.05）and the content of SFA decreased;the proportion of C16:1,C18:1 decreased significantly（P<0.01）,and the content of MUFA decreased.Meanwhile,the proportion of C18:2,AA and EPA decreased significantly（P<0.01）,and the content of PUFA decreased.Bodipy staining showed that the lipid droplets in bovine adipocytes decreased after the ACSL1 gene was silenced.In summary,overexpression and interference of ACSL1 gene play an important role in regulating the expression of genes related to unsaturated fatty acid synthesis,fatty acid composition and lipid droplet formation in bovine adipocytes,which provides a theoretical basis for the follow-up study of the regulation mechanism of unsaturated fatty acid deposition in beef and the molecular mechanism of unsaturated fatty acid composition in bovine adipocytes.