The Cloning And Expression Of ACSL1 And Its Effect On Fatty Content

Long-chain fatty acyl-Co A synthetases(ACSLs)is a kind of enzyme which is much important for activation of fatty acid in mammal. ACSLs could transform fatty acid to Ester acyl coenzyme A. The results of the indicate that ACSLs not only play a key role in the synthesis of triglycerides, phospholipids and cholesterol, but also affect the metabolism of fatty acid.According to the predictions of ACSL1 gene sequence published in Genebank, we used the public sequence as the target to design primers. The results showed that the sequence length of ACSL1 was 2100 bp encoding one opening reading fram with 699 amino acid residues. The molecular weight of ACSL1 was 82.76 k D, and the theoretical p I was 5.38. ACSL1 was a hydrophobic protein without signal peptide, and not a secreted protein.Compared with that of pig and cattle, the homology of amino acids reached 97%.Using Real- time fluorescence quantitative PCR, the expression differences of ACSL1 gene in various tissues of sheep were investigred, showing that the expression of ACSL1 gene was different in various tissues. The expression quantity followed the order of liver, heart, subcutaneous fat, duodenum, kidneys, lung, spleen and testis from high to low.The molecular cloning technology was used to build the RNA interference vector and expression vector of ACSL1 gene, that were then mediated into sheep fatty cells by liposome transfection. Screening for genetically modified cells by G418, we utilized Real- time fluorescence quantitative PCR to test the change of m RNA, finding that expression vector and RNA interferenceⅠtranscription and regulation the ACSL1 gene effectively. Using triglycerides kit to test the triglyceride content in the cell, the results showed that ACSL1 gene may play an important role in regulating animal fatty metabolism.Contrasting with amplified sequence of ACSL1, one mutational site was found at the exon 2 of ACSL1 gene, which was chr.26 13930064 G-A mutation. One mutational site was found at the exon 14 of ACSL1 gene, which was chr.26 13929965 C-T mutation.There was also one mutational site at the exon18, which was chr.26 13922514 G-A mutation. Five mutational sites were found at the intron of ACSL1 gene. We will analyse the association of ACSL1 gene polymorphism with meat traits. The results showing that exon2 of ACSL1 gene had an effect on the content of fatty acid and amino, and exon14 on amino and nutrition.