Effect Of ACAA1 Gene On Lipid Metabolism And Analysis Of Genetic Polymorphism In Sheep

Acetyl-CoA acyltransferase 1(ACAA1)is a member of the thiolytic enzyme family,also known as peroxisome 3-ketoacyl-CoA thiolytic enzyme.It is mainly involved in the physiological and biochemical processes such as β-oxidation of very long-chain fatty acids,long-chain fatty acids and branched-chain fatty acids,regulation of peroxisome proliferation and bile acid metabolism,and it is the key regulatory enzyme of fatty acid β-oxidation in peroxisomes.Through catalyticβ-oxidation,3-ketoacyl-CoA is cleaved to acetyl-CoA and acyl-CoA in peroxisome to acetyl-CoA and acyl-CoA,which participate in the extension and degradation of fatty acids.It is an important regulatory gene in the process of lipid metabolism and plays a key role in the process of fatty acid oxidation and lipid metabolism.ACAA1 can regulate fatty acid oxidation in PPAR signaling pathway and participate in the maintenance of lipid balance.The oxidation of fatty acids means the change of fatty acid content,which may affect the meat quality of livestock and poultry.To explore the regulation effect of ACAA1 gene on lipid metabolism and its genetic polymorphism in sheep.In this experiment,ACAA1 was used as the research object,the CDS region of ACAA1 gene was cloned by RT-PCR,and its bioinformatics and tissue expression differences were analyzed in order to understand its structure and predict its function.Then,overexpression vector and siRNA interference of sheep ACAA1 gene were constructed and transfected into sheep preadipocytes,then the differentiation was induced,and RNA and protein were extracted from different induction periods cells.qRT-RCR and Western blot were used to detect the effects of overexpression and knockdown of ACAA1 gene on the differentiation of sheep preadipocytes at the level of mRNA and protein,respectively.Finally,the polymorphism of this gene was detected by PCR product Sanger sequencing in sheep population,and the linkage analysis between SNPs and meat quality traits was carried out,in order to screen out molecular markers related to meat quality traits of sheep,and provide reference basis for genetic improvement and breeding of mutton sheep in the future.The main results are as follows:1.Cloning,bioinformatics and tissue expression profile analysis of ACAA1 gene CDS in sheepThe full-length coding sequence of ACAA1 gene of small-tailed Han sheep was cloned successfully,which encodes 356 amino acids with a theoretical isoelectric point of 7.48,which is an unstable alkaline fat-soluble hydrophilic protein.ACAA1 protein has no transmembrane structure and signal peptide,and contains 25 phosphorylation sites,3 N-glycosylation sites and 4 O-glycosylation sites.Its secondary structure contains α helix(37.92%),β-sheet(12.64%)and irregular crimp(49.44%),which is consistent with the predicted tertiary structure.ACAA1 gene was expressed in different tissues of small tail Han sheep,and the expression level was relatively high in liver,subcutaneous fat and small intestine.2.Effect of ACAA1 gene on preadipocytes differentiation in sheep(1)Sheep preadipocytes were successfully isolated and nduced differentiation system was established in vitro.(2)Expression of ACAA1 gene was detected during the differentiation of sheep preadipocytes.It was found that the expression of ACAA1 increased in the early stage of differentiation and decreased gradually with the process of differentiation.The expression trend of mRNA and protein was similar to that of adipogenic marker gene C/EBP α.(3)Expression of ACAA1 was regulated in preadipocytes and its effect on adipocyte differentiation was explored.It was found that overexpression of ACAA1 gene could reduce the number of lipid droplets and triglyceride content in mature adipocytes,and down-regulated the mRNA and protein expression of adipogenic marker genes PPAR y and C/EBP a during adipogenic differentiation.On the contrary,interfering with ACAA1 gene increased lipid droplet accumulation and triglyceride content,and up-regulated the mRNA and protein expression of PPARγ and C/EBP α.3.Detection of genetic polymorphism of ACAA1 gene and analysis of its association with meat quality traits in sheep.The SNP loci of ACAA1 gene were screened in the hybrid sheep population of Duper sheep and small tail Han sheep.The results showed that among the 14 exons of ACAA1 gene,only g.1434 A>G was found in the 3 ’untranslated region(UTR)of exon 14,and g.1484 C>T was found in the 3 ’flanking sequence.The association analysis of different genotypes combined with meat quality traits showed that the three genotypes of A/G locus were significantly correlated with drip loss(P<0.05),and 3 ’flanking sequence genotypes were significantly correlated with water loss rate and drip loss(P<0.05).There was no significant difference in other indexes(P>0.05).In summary,the coding sequence of ACAA1 gene of small-tailed Han sheep was cloned successfully,and the difference of gene expression in different tissues of small-tailed Han sheep was confirmed.In the process of adipogenic differentiation of sheep preadipocytes,interference with ACAA1 promotes preadipocyte differentiation,and overexpression of ACAA1 inhibits preadipocyte differentiation.This gene may be a negative regulator of adipocyte differentiation.The two SNP loci of ACAA1 gene in sheep were correlated with drip loss,water loss rate and some meat quality traits.This study provides a theoretical basis and reference for further exploring the function of gene and the breeding of meat quality traits in sheep.