暗纹东方鲀心型脂肪酸结合蛋白H-FABP基因克隆与表达分析

为深入了解心型脂肪酸结合蛋白(heart-type fatty acid-binding protein,H-FABP)的结构与功能,采用RACE技术克隆得到暗纹东方鲀H-FABP的全长cDNA序列,共772 bp,开放阅读框为402 bp,编码133个氨基酸。生物信息学分析表明,暗纹东方鲀H-FABP基因具有一个脂质转运蛋白家族保守结构域和多个蛋白激酶磷酸化位点。NJ法系统进化分析显示,暗纹东方鲀与红鳍东方鲀亲缘关系最近,其次是鲈形目。经荧光定量PCR检测,H-FABP基因在暗纹东方鲀所有被检测的组织中都有表达,其中肝脏中的表达量最高,其次为肌肉和心脏,预示H-FABP主要参与脂肪酸氧化分解过程。为探究豆油作为脂肪源的可行性,采用不同比例(0、25%、50%和75%)豆油替代鱼油的饲料投喂初始体质量为(8.31±0.20)g的暗纹东方鲀8周。结果显示,全鱼油组肝脏中H-FABP基因的表达量显著高于50%豆油组和75%豆油组,但与25%豆油组无显著性差异,且随着饲料中豆油比例的增加,暗纹东方鲀肝脏中H-FABP基因的表达量呈下降趋势。研究表明,富含n-3多不饱和脂肪酸的鱼油可以促进脂肪酸氧化分解,能预防脂肪肝。因此,为不影响暗纹东方鲀肝脏脂肪代谢,饲料中豆油替代鱼油的比例应控制在50%以下。

Molecular cloning and feature analysis of heart-type fatty acid-binding protein H-FABP gene from obscure puffer(Takifugu obscurus)

In order to understand the structure and function of heart type fatty acid binding protein H-FABP,the full-length cDNA sequence of H-FABP gene was cloned from obscure puffer(Takifugu obscurus)by using SMART RACE technique.The entire H-FABP cDNA was 772 bp,containing a 402 bp open reading frame encoding a protein with 133 amino acids.Bioinformatics analysis indicated that the H-FABP gene had a conserved domain of lipocalin family and several phosphorylation sites.Phylogenetic analysis indicated that the H-FABP gene of Takifugu obscurus was closely related to Takifugu rubripes,followed by that of Perciformes.Real-time PCR revealed that H-FABP gene was ubiquitously expressed in all tested tissues.The highest expression of H-FABP was tested in the liver,followed by muscle and heart.This indicates that H-FABP is mainly involved in fatty acid oxidation process.To evaluate the feasibility of using soybean oil as a dietary lipid,juvenile fish were fed with four formulated diets differing in lipid composition,with soybean oil accounting for 0,25%,50% and 75% of total dietary lipid respectively.After feeding for 8 weeks,the expression of H-FABP gene in the liver of the fish oil group was significantly higher than those in 50% soybean oil group and 75% soybean oil group(P<0.05).However,there was no significant difference(P >0.05)between the 25% soybean oil group and the fish oil group.The expression of H-FABP gene in the liver decreased with the increasing of the proportion of soybean oil in diets,indicating that fish oil rich in n-3 polyunsaturated fatty acids can promote fatty acid oxidation and prevent fatty liver.Therefore,in order not to affect the liver fat metabolism of obscure puffer,the proportion of soybean oil instead of fish oil should be less than 50%.