Study On The Associations Of Duck L-FABP And A-FABP Gene Polymorphisms With Intramuscular Fat Contents And Their Influences On The Expression Of Lipid-metabolism Relared Genes

Fatty acid-binding proteins (FABPs) are members of a family of intracellular lipid-binding proteins (iLBPs) involved in the transportation of fatty acids, especially long-chain fatty acids, from plasma membrane to the sites of β oxidation and/or triacylglycerol or phospholipids synthesis. The molecular weights of FABPs are12-15kDa. Different types of FABPs function in different tissues. In this study, we obtained the gene sequences of liver-type and adipocyte-type FABPs, detected the SNPs in the exons of the two genes, and analyzed their associations with the IMF content and the fatty acids ratios in duck. We also detected the expression patterns of the two FABPs in various tissues by Real-time PCR, and studied the regulatory mechanism of L-FABP gene in hepatocytes and A-FABP gene in adipocytes in duck with RNAi technique. The results are as follows:1. Duck L-FABP and A-FABP gene sequencesDuck L-FABP and A-FABP gene sequences were acquired by gene cloning and sequencing. The obtained duck L-FABP gene sequence is2,542bp in length, including4exons and3introns, and the CDS regions are52â†'118bp,1,113â†'1,285bp,1,581â†'1670bp, and2,421â†'2,471bp, respectively, encoding126amino acids, which has been submitted to GenBank (Accession No.:HQ640427). The obtained duck L-FABP gene sequence is3,338bp, including4exons,3introns and partial5’ flanking region, and the CDS regions are205â†'277bp,1,539â†'1,711bp,1,929â†'2,030bp,3,288â†'3,338bp, encoding132amino acids, which has been submitted to GenBank (Accession No.:HQ640428)2. Detection of the SNPs and their associations with IMF and fatty acid contentsOne SNP was found in the exon3of duck L-FABP gene:HQ640427:g.1953C>T, and its polymorphism was proven to be associated with C16:0, C18:3and IMF in duck pectoral muscle:Two SNPs were detected in the exon3of duck A-FABP Gene:HQ640428:g.1991T>C and HQ640428:g.2018A>G, the polymorphism of HQ640428:g.2018A>G was determined to be associated with the contents of C18:0, C18:2, C18:3and total IMF in duck pectoral muscle. No linkage was detected between the SNPs.3. Expression patterns of duck L-FABP and A-FABP genes in various tissuesReal-time PCR was performed to detect the expression patterns of duck L-FABP and A-FABP genes in15tissues. The results showed that duck L-FABP was highly expressed in liver, also expressed in various tissues except pancreas and spleen; duck A-FABP was highly expressed in fat tissues, also expressed in muscle and other tissues except pancreas, lung and kidney.4. The regulations of duck L-FABP gene in the hepatocytes and A-FABP gene in the adipocytesThe RNAi technique was used to knockdown L-FABP gene in hepatocytes and A-FABP gene in adipocytes in duck to study its regulations to the lipid-metabolism related genes. The L-FABP gene knockdown inhibited its mRNA expression by58.7%. meanwhile, the mRNA expression of FAS, LPL and PPARa genes were also significantly decreased in the hepatocytes, and no significant difference was determined on LEPR or PPARy gene expression. The A-FABP gene knockdown resulted in77.0%decrease in its mRNA expression in the duck adipocytes, and the expression of FAS, LEPR, LPL and PPARγ genes were also down-regulated, but no significant inhibition was detected on PPARa gene expression. The stimulation of oleic acid could relieve both the suppressions. These results indicate that the two genes play important roles in regulating the expression of fat-related genes.In this study, we found that polymorphisms of duck L-FABP and A-FABP genes are associated with IMF contents. Both the two gene are expressed in various tissues, and could regulate the expression levels of lipid metabolism related genes in hepatocytes and adipocytes, respectively.