AGPAT4基因干扰及过表达对西农萨能奶山羊乳腺上皮细胞脂代谢的影响  

Effects of AGPAT4 Gene Interference and Overexpression on Lipid Metabolism in Mammary Epithelial Cells of Xinong Saanen Dairy Goats(Capra hircus)

作　　者：姚玮玮 罗军[1] 王馨培 屈小鹏 田慧彬 牛慧敏 YAO Wei-Wei;LUO Jun;WANG Xin-Pei;QU Xiao-Peng;TIAN Hui-Bin;NIU Hui-Min(College of Animal Science and Technology,Northwest A&F University/Shaanxi Provincial Key Laboratory of Molecular Biology for Agriculture,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物科技学院/陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2023年第9期1868-1877,共10页Journal of Agricultural Biotechnology

基　　金：国家科技重大专项转基因新品种培育(2018ZX0800802B);国家自然科学基金(31772575)。

摘　　要：溶血磷脂酸酰基转移酶4(1-acylglycerol-3-phosphate O-acyltransferase 4, AGPAT4)是1-酰基甘油-3-磷酸O酰基转移酶家族的成员,具有催化甘油三酯合成的作用。为了阐明AGPAT4在山羊(Capra hircus)乳腺上皮细胞(goat mammary epithelial cells, GMECs)中的基因序列、结构以及AGPAT4对GMECs脂肪酸合成代谢的影响。本研究以西农萨能奶山羊乳腺上皮细胞为实验材料,通过RT-PCR克隆AGPAT4基因,应用在线网站进行生物信息学分析,干扰与过表达AGPAT4进行功能分析。结果表明,本研究成功克隆得到AGPAT4基因CDS区1 137 bp(sequence ID:XM_005684959.3),编码378个氨基酸,蛋白分子量为43 939.35 u,理论pI值为8.92,存在跨膜结构,无信号肽,为携带正电荷的不稳定蛋白;AGPAT4蛋白与AGPAT1、AGPAT2、脂蛋白3(lipin3, LPIN3)、二酰基甘油激酶(diacylglycerol kinases, DGK)、磷脂酶D(phospholipase D, PLD)、胞苷二磷酸-二酰基甘油合成酶(cytidine diphosphate-diacylglycerol synthase,CDS)等蛋白存在互作关系;干扰AGPAT4显著下调乙酰辅酶A羧化酶(acetyl coa carboxylase, ACC)基因表达(P<0.05);过表达AGPAT4显著上调硬脂酰辅酶A去饱和酶1(stearoyl-coenzyme A desaturase, SCD1)与ACC基因表达(P<0.05)并显著促进细胞内甘油三酯积累(P<0.05)。以上结果表明,AGPAT4基因在山羊乳腺上皮细胞中对脂合成具有正向调控作用。本研究为后续开展AGPAT4基因对羊奶乳脂代谢的调控机制研究提供了基础资料。AGPAT4(1-acylglycerol-3-phosphate O-acyltransferase 4)is a member of the 1-acylglycerol-3-phosphate O acyltransferase family of enzymes that catalyze synthesis of the triacylglycerol(TAG).In order to clarify the gene sequence and protein structure of AGPAT4 and to preliminarily analyze the effect of AGPAT4 on lipid metabolism in goat mammary epithelial cells(GMECs),In this study,the mammary epithelial cells of Xinong Saanen dairy goat(Capra hircus)were used as experimental materials,the AGPAT4 gene was cloned by reverse transcription-PCR(RT-PCR).Based on the cloned sequence,the bioinformatics analysis was conducted using online website and tools.qPCR was applied to detect the expression of GMECs lipid metabolism-related genes after AGPAT4 interference and overexpression.Then,the content of triacylglycerol in GMECs was detected after interference or overexpression of AGPAT4.The results showed that the CDS region of AGPAT4 gene was successfully cloned,with 1137 bp(sequence ID:XM_005684959.3),encoding 378 amino acids,protein molecular weight of 43939.35 u,theoretical pI value of 8.92,transmembrane structure and no signal peptide,which was an unstable protein with positive charge.The AGPAT4 protein interacted with AGPAT1,AGPAT2,lipin3(LPIN3),diacylglycerol kinases(DGK),phospholipase D(PLD),cytidine diphosphate-diacylglycerol synthase(CDS)and other proteins.The downregulation of AGPAT4 via mRNA interference caused a significant reduction in acetyl coa carboxylase(ACC)gene expression(P<0.05).Overexpression of AGPAT4 significantly up-regulated the expression of stearoyl-coenzyme A desaturase(SCD1)and ACC genes(P<0.05)and significantly promoted the accumulation of intracellular TAG(P<0.05).These results indicated that AGPAT4 gene had positive regulation on lipid synthesis in GMECs.This study provides basic data for further research on the regulatory mechanism of AGPAT4 gene on milk fat metabolism of goat milk.

关 键 词：西农萨能奶山羊 溶血磷脂酸酰基转移酶4(AGPAT4) 脂代谢 山羊乳腺上皮细胞(GMECs) 

分 类 号：S827[农业科学—畜牧学]