奶山羊ACSL6基因克隆及表达调控分析

研究旨在获得西农萨能奶山羊长链脂酰CoA合成酶6（long-chain acyl-CoA synthetase 6,ACSL6）基因CDS序列,初步探究其对奶山羊乳腺上皮细胞脂质代谢的影响。以西农萨能奶山羊原代乳腺上皮细胞为试验材料,对ACSL6基因进行扩增和克隆,并利用实时荧光定量PCR方法对ACSL6基因进行组织表达分析,针对ACSL6基因的碱基序列利用在线软件进行生物信息学分析,采用RNA干扰技术改变奶山羊乳腺上皮细胞中ACSL6基因mRNA的表达水平。结果显示,ACSL6基因CDS区全长为2 169 bp,编码722个氨基酸;生物信息学分析表明ACSL6蛋白为碱性不稳定蛋白。组织表达分析显示,ACSL6基因在奶山羊乳腺组织中高度表达,其次为脾脏。将设计合成的siRNA转染乳腺上皮细胞,发现干扰ACSL6基因的表达使脂质代谢相关基因乙酰辅酶A羧化酶（acetyl CoA carboxylase,ACC）、硬脂酰辅酶A去饱和酶1（stearyl-CoA dehydrogenase 1,SCD1）、脂肪酸转运蛋白36（cluster of differentiation 36,CD36）、固醇调节元件结合蛋白1（sterol regulatory element binding proteins 1,SREBP1）的mRNA表达水平极显著下调（P<0.01）。本研究结果为从蛋白及个体水平上研究ACSL6基因对奶山羊乳腺脂质代谢的影响提供理论依据。

Cloning and Expression Regulation Analysis of ACSL6 Gene in Dairy Goats

The aim of this study was to obtain the coding sequences of long-chain acyl-CoA synthetase 6 (ACSL6) gene in Xinong Saanen dairy goats,and preliminarily explore its effect on lipid metabolism of mammary epithelial cells in dairy goats.Taking the primary mammary epithelial cells of Xinong Saanen dairy goats as experimental materials,ACSL6 gene was amplified and cloned,and tissue expression profiles were analyzed by Real-time quantitative PCR,bioinformatics analysis was carried out based on the sequence of ACSL6 gene by online software,RNA interference technology was applied to interfere the mRNA expression of ACSL6 gene in mammary epithelial cells of dairy goats.The results showed that the length of ACSL6 gene CDS region was 2 169 bp,encoded 722 amino acids.Bioinformatics analysis indicated that ACSL6 protein was an alkaline unstable protein.The results of tissue expression analysis showed that ACSL6 gene was highly expressed in mammary tissue,followed by spleen in dairy goats.The designed synthetic siRNA was transfected into mammary epithelial cells,interference of ACSL6 gene made the mRNA expression of acetyl-CoA carboxylase (ACC),stearyl-CoA dehydrogenase 1 (SCD1),cluster of differentiation 36 (CD36) and sterol regulatory element binding proteins 1 (SREBP1) genes extremely significantly decreased (P<0.01).This results laid the foundation for further study on the effect of ACSL6 gene on lipid metabolism at the protein level and individual level.