奶山羊ACSL6基因启动子克隆与转录活性分析  

Cloning and Transcriptional Activity Analysis of ACSL6 Gene Promoter in Dairy Goats (Capra hircus)

作　　者：熊畑畑 吴钰 邵钺馨 陈冲 王琬婷 程菲 李聪 XIONG Tian-Tian;WU Yu;SHAO Yue-Xin;CHEN Chong;WANG Wan-Ting;CHENG Fei;LI Cong(College of Animal Science and Technology,Northwest A&F University/Shaanxi Provincial Key Laboratory of Animal Genetics,Breeding and Reproduction,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物科技学院/陕西省动物遗传育种与繁殖重点实验室,杨凌712100

出　　处：《农业生物技术学报》2025年第1期124-133,共10页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2022YFD1300203);陕西省畜禽育种“两链”融合重点专项(2022GD-TSLD-46-0203)。

摘　　要：酰基辅酶A合成酶长链家族成员6(acyl-CoA synthetase long chain family member 6, ACSL6)作为摄取、转运游离脂肪酸的关键酶,参与调控奶畜泌乳中脂肪酸的代谢过程。本研究旨在通过对奶山羊(Capra hircus) ACSL6基因(Gene ID:XM_018050725.1)启动子克隆、生物信息学分析和双荧光素酶活性检测,初步探究ACSL6基因的转录调控机制。以西农萨能奶山羊血液基因组DNA为模板,经PCR扩增获得ACSL6启动子的全长序列;利用生物信息学软件筛选预测分值较高的结合位点和启动子特征序列;克隆并构建启动子缺失片段载体,与内参载体共同转染奶山羊乳腺上皮细胞,通过双荧光素酶活性检测确定启动子活性区域。结果表明,克隆获得ACSL6基因启动子全长序列2 108 bp;ACSL6启动子上存在多种转录因子的结合位点;在启动子序列-907~-807 bp和-513~-54 bp存在2个长度分别为101和460 bp的CpG岛;对克隆获得的9个缺失片段进行双荧光素酶活性分析,发现ACSL6基因启动子的核心区域定位于-170~-33 bp,在转录起始位点上游-2 004~-1 707 bp、-1 334~-957 bp和-584~-271 bp可能存在负调控元件。本研究为揭示ACSL6调控奶山羊乳脂代谢的分子机理提供基础资料,为从分子层面改善羊乳品质提供参考。Acyl-CoA synthetase long chain family member 6(ACSL6),a key enzyme in the uptake and transport of free fatty acids,is involved in the regulation of fatty acid metabolism during lactation in dairy animals.This study aimed to preliminarily investigate the transcriptional regulation mechanism of ACSL6 gene(Gene ID:XM_018050725.1)through promoter cloning,bioinformatics analysis and diluciferase activity detection in dairy goats(Capra hircus).The full-length sequence of the ACSL6 promoter was obtained by PCR amplification using the blood genomic DNA of Xinong Saanen dairy goats as a template.The sequence features of the binding sites and promoter regions with high prediction scores were screened using bioinformatics analysis software.The promoter deletion fragment vectors were cloned and constructed,which were co-transfected with the internal reference vectors into the mammary epithelial cells of dairy goats,and the promoter active regions were identified by dual luciferase activity assay.The results showed that the full-length sequence of the ACSL6 promoter was 2108 bp.The multiple transcription factor binding sites were existed in the ACSL6 promoter.The two CpG islands with lengths of 101 and 460 bp were identified in the promoter sequences of-907~-807 bp and-513~-54 bp,respectively.The nine deletions obtained from the cloning were analysed by dual-luciferase activity,and it was found that the core region of the ACSL6 promoter was located at-170~-33 bp,and there might be negative regulatory elements upstream of the transcriptional start site at-2004~-1707 bp,-1334~-957 bp,and-584~-271 bp.This study provides basic information to reveal the molecular mechanism of ACSL6 regulating milk fat metabolism in dairy goats,and provides a reference to improve the quality of goat milk at the molecular level.

关 键 词：酰基辅酶A合成酶长链家族成员6(ACSL6) 启动子克隆 片段缺失分析 生物信息学分析 转录因子 

分 类 号：S813.3[农业科学—畜牧学] S826.94[农业科学—畜牧兽医] S827