鸡PGC-1α基因真核表达载体的构建及其对脂类代谢的作用  

Construction of Eukaryotic Expression Vector for Chicken(Gallus gallus)PGC-1αGene and Its Effect on Lipid Metabolism

作　　者：周博 魏志恒 刘世昊 刘书鸣 夏磊 徐璐 郁建锋[1] 顾志良[1] ZHOU Bo;WEI Zhi-Heng;LIU Shi-Hao;LIU Shu-Ming;XIA Lei;XU Lu;YU Jian-Feng;GU Zhi-Liang(School of Biology and Food Engineering,Changshu Institute of Technology,Changshu 215500,China)

机构地区：[1]常熟理工学院生物与食品工程学院,常熟215500

出　　处：《农业生物技术学报》2025年第2期344-354,共11页Journal of Agricultural Biotechnology

基　　金：江苏省自然科学基金(BK20191476);常熟理工学院2023大学生创业创新计划资助。

摘　　要：过氧化物酶体增殖物激活受体γ共激活因子-1α(peroxisome proliferator activated receptorγcoactivator-1 alpha,PGC-1α)是一种转录共激活因子,参与调控体内各类能量代谢过程,对脂肪沉积起着关键作用。为探讨PGC-1α基因在鸡(Gallus gallus)脂质代谢中的作用和机制,本研究检测了PGC-1α基因在鸡12种组织中的表达情况,通过RT-PCR的方法,扩增并获得鸡PGC-1α基因CDS片段,构建真核表达载体,Western blot检测表达载体转染鸡肝癌细胞系(leghorn male hepatoma,LMH)后能否表达PGC-1α蛋白;过表达和敲低LMH细胞中PGC-1α基因后,油红O染色检测脂滴沉积变化,比色法测定总甘油三酯(total triglyceride,TG)含量的变化,qPCR检测对脂质代谢相关基因表达的影响。结果显示,PGC-1α基因在心脏组织中表达量最高,在腿肌、肾和脑组织中表达量较高;Western blot检测发现构建的过表达载体转染LMH细胞能成功表达融合蛋白;LMH细胞中过表达PGC-1α基因会使脂质沉积和TG含量极显著下降(P<0.01),而PGC-1α基因敲低后脂质沉积有增加趋势,且TG含量显著增加(P<0.05)。LMH细胞中PGC-1α基因过表达使得脂肪酸合成酶(fatty acid synthetase,FASN)基因和过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorγ,PPARγ)基因的表达极显著下调(P<0.01),乙酰辅酶A羧化酶α(acetyl-CoA carboxylase alpha,ACACA)基因的表达显著下调(P<0.05),磷酸烯醇式丙酮酸羧激酶1(phosphoenolpyruvate carboxykinase 1,PCK1)基因表达极显著上调(P<0.01);PGC-1α基因敲低使PCK1基因表达极显著下降(P<0.01)。上述结果表明,鸡PGC-1α基因可能是通过调节上述功能基因表达而发挥作用。本研究通过构建PGC-1α基因的过表达和敲低模型,揭示了该基因在鸡脂质代谢中的关键调控作用,尤其是通过调节FASN、PPARγ、ACACA和PCK1等基因的表达,影响脂质沉积和甘油三酯含量,表明PGC-1α基因可能在调控鸡体内脂肪代�Peroxisome proliferator activated receptorγcoactivator-1 alpha(PGC-1α)is a transcriptional coactivator that influences various energy metabolism processes and plays a crucial role in fat deposition.To investigate the function and underlying mechanisms of the PGC-1αgene in lipid metabolism,a series of experiments were performed.The expression of PGC-1αin 12 different tissues was examined in chickens(Gallus gallus).Using RT-PCR,the CDS of the chicken PGC-1αgene was amplified and obtained,and a eukaryotic expression vector was constructed.The vector was then transfected into the leghorn male hepatoma(LMH)cell line,and Western blot analysis was used to confirm the expression of the PGC-1αprotein.After overexpressing and knocking down PGC-1αgene in LMH cells,lipid droplet deposition was assessed using oil red O staining,total triglyceride(TG)content was measured by colorimetric assay,and the expression of lipid metabolism-related genes was analyzed through qPCR.The results showed that PGC-1αhad the highest expression in cardiac tissue,with relatively high levels in leg muscle,kidney,and brain tissues.Western blot analysis confirmed that the overexpression vector successfully expressed the fusion protein in LMH cells.Overexpression of PGC-1αled to a extremely significant reduction in lipid deposition and TG content(P<0.01),while knockdown of PGC-1αresulted in a trend of increased lipid deposition and a significant rise in TG content(P<0.05).Moreover,overexpression of PGC-1αsignificantly downregulated the expression of fatty acid synthetase(FASN),peroxisome proliferator activated receptorγ(PPARγ)and acetyl-CoA carboxylase alpha(ACACA)genes(P<0.05 or P<0.01),while extremely significantly upregulated phosphoenolpyruvate carboxykinase 1(PCK1)(P<0.01).Knockdown of PGC-1αextremely significantly decreased PCK1 expression(P<0.01).These findings suggested that the chicken PGC-1αgene likely exerts its effects by regulating the expression of these functional genes.By constructing overexpression and knockdown models

关 键 词：鸡 过氧化物酶体增殖物激活受体γ共激活因子-1α(PGC-1α)基因 真核表达 鸡肝癌细胞系(LMH)细胞 脂代谢 脂肪沉积 

分 类 号：S831.2[农业科学—畜牧学]