鸭L-FABP、A-FABP基因多态性与肌内脂肪含量的相关性及其对脂肪代谢相关基因表达的影响
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摘要
脂肪酸结合蛋白(FABPs)是细胞内脂质结合蛋白(iLBs)中的一类成员。FABPs是一类分子量约12-15kDa勺细胞溶质蛋白,对长链脂肪酸有高度的亲和性,在动物体内对脂肪酸和它们的CoA衍生物的摄取、胞内转运、脂化、氧化以及合成均有重要作用。不同类型的FABPs在不同的组织中发挥生物功能。本试验选取鸭肝脏型L-FABP口脂肪细胞型A-FABP,克隆其基因序列,检测外显子上的SNPs,并分析其与肌内脂肪IMF含量的相关性。用荧光定量方法检测该两基因在不同组织中的表达情况,并运用RNAi方法分别研究其在鸭肝细胞和脂肪细胞中对脂肪代谢相关基因的调控作用.以及油酸对该调控作用的影响。主要结果如下
J.鸭L-FABP与A-FABP基因序列的获得
经克隆测序得到了鸭L-FABP与A-FABP基因的序列。获得的鸭L-FABP基因序列长度为2.542bp,包含4个外显子和3个内含子,其中CDS序列分别为52→118bp,1,13→1.285bp、1,581→1670bp,2,421→2,471bp,共编码126个氨基酸,此结果已上传GenBank (Accession No.:HQ640427)获得的鸭A-FABP基因呼列长度为3,338bp.包含4个外显子和3个内含子以及部分5’端序列,其中CDS序列分别为205→277bp,1,539→1,711bp、1,929→2,030bp,3,288→3,338bp,共编码132个氨基酸,此结果已上传GenBank (Accession No.:HQ640428)。
2. SNPs的检测及其与IMF含量的相关性分析
在鸭L-FABP外显子3上发现1个SNP:HQ640427:g.1953C>T,并发现其与胸肌的IMF含量以及其中棕榈酸和亚麻酸的含量呈显著相关性；在鸭A-FABP基因的外显子3上发现2个SNPs:HQ640428:g.1991T>C和HQ640428:g.2018A>G,其中HQ640428:g.2018A>G的多态性与胸肌IMF含量以及其中硬脂酸、亚油酸和亚麻酸的含量呈显著相关性。本试验检测到的SNPs之间无连锁交互关系。
3.鸭L-FABP和A-FABP基因在各组织中的表达丰度
应用荧光定量PCR法检测了鸭L-FABP和A-FABP基因在所选的15个组织中的表达情况。发现鸭L-FABP基因在肝脏中高度表达,且在除胰腺和脾脏外的其它多种组织中均有表达；鸭A-FABP基因在脂肪组织中高度表达,且在除胰腺、肺和肾脏外的其它多种组织中均有表达。
4.鸭L-FABP在肝细胞及A-FABP在脂肪细胞中的调控作用
本试验用RNAi方法对鸭肝细胞L-FABP基因进行knockdown,发现其表达量降低了58.7%,同时FAS、LPL和PPARa基因的表达量也均显著下调,油酸的刺激可以缓解这种抑制作用,但LEPR和PPARy表达量无显著变化；当对鸭脂肪细胞A-FABP基因进行knockdown后,发现其表达量降低了77.0%,FAS、LEPR、LPL和PPARy基因的表达量也均显著下调,油酸的刺激可以缓解这种抑制作用,但PPARa基因表达量却无显著变化。这表明该两基因对脂肪相关基因的表达具有一定的调控作用。
从结果中可以看出,鸭L-FABP和A-FABP基因多态性均与胸肌脂肪含量具有显著相关性,在其他多种组织均有表达,且在脂肪代谢中具有重要的调控作用,能影响多种脂肪代谢相关基因的表达。
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 sequences
Duck 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 are52→118bp,1,113→1,285bp,1,581→1670bp, and2,421→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 are205→277bp,1,539→1,711bp,1,929→2,030bp,3,288→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 contents
One 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 tissues
Real-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 adipocytes
The 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.
J.鸭L-FABP与A-FABP基因序列的获得
经克隆测序得到了鸭L-FABP与A-FABP基因的序列。获得的鸭L-FABP基因序列长度为2.542bp,包含4个外显子和3个内含子,其中CDS序列分别为52→118bp,1,13→1.285bp、1,581→1670bp,2,421→2,471bp,共编码126个氨基酸,此结果已上传GenBank (Accession No.:HQ640427)获得的鸭A-FABP基因呼列长度为3,338bp.包含4个外显子和3个内含子以及部分5’端序列,其中CDS序列分别为205→277bp,1,539→1,711bp、1,929→2,030bp,3,288→3,338bp,共编码132个氨基酸,此结果已上传GenBank (Accession No.:HQ640428)。
2. SNPs的检测及其与IMF含量的相关性分析
在鸭L-FABP外显子3上发现1个SNP:HQ640427:g.1953C>T,并发现其与胸肌的IMF含量以及其中棕榈酸和亚麻酸的含量呈显著相关性；在鸭A-FABP基因的外显子3上发现2个SNPs:HQ640428:g.1991T>C和HQ640428:g.2018A>G,其中HQ640428:g.2018A>G的多态性与胸肌IMF含量以及其中硬脂酸、亚油酸和亚麻酸的含量呈显著相关性。本试验检测到的SNPs之间无连锁交互关系。
3.鸭L-FABP和A-FABP基因在各组织中的表达丰度
应用荧光定量PCR法检测了鸭L-FABP和A-FABP基因在所选的15个组织中的表达情况。发现鸭L-FABP基因在肝脏中高度表达,且在除胰腺和脾脏外的其它多种组织中均有表达；鸭A-FABP基因在脂肪组织中高度表达,且在除胰腺、肺和肾脏外的其它多种组织中均有表达。
4.鸭L-FABP在肝细胞及A-FABP在脂肪细胞中的调控作用
本试验用RNAi方法对鸭肝细胞L-FABP基因进行knockdown,发现其表达量降低了58.7%,同时FAS、LPL和PPARa基因的表达量也均显著下调,油酸的刺激可以缓解这种抑制作用,但LEPR和PPARy表达量无显著变化；当对鸭脂肪细胞A-FABP基因进行knockdown后,发现其表达量降低了77.0%,FAS、LEPR、LPL和PPARy基因的表达量也均显著下调,油酸的刺激可以缓解这种抑制作用,但PPARa基因表达量却无显著变化。这表明该两基因对脂肪相关基因的表达具有一定的调控作用。
从结果中可以看出,鸭L-FABP和A-FABP基因多态性均与胸肌脂肪含量具有显著相关性,在其他多种组织均有表达,且在脂肪代谢中具有重要的调控作用,能影响多种脂肪代谢相关基因的表达。
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 sequences
Duck 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 are52→118bp,1,113→1,285bp,1,581→1670bp, and2,421→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 are205→277bp,1,539→1,711bp,1,929→2,030bp,3,288→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 contents
One 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 tissues
Real-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 adipocytes
The 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.
引文
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