奶山羊心脏型脂肪酸结合蛋白(FABP3)基因启动子的克隆及活性分析  

Cloning and activity analysis of the promoter of the heart fatty acid binding protein(FABP3)gene in dairy goats

作　　者：姚大为[1] 赵欣 赵淑颖 杨春蕾[1] 李玉鹏[1] 马毅[1] YAO Dawei;ZHAO Xin;ZHAO Shuying;YANG Chunlei;LI Yupeng;MA Yi(Institute of Animal Husbandry and Veterinary Science,Tianjin Academy of Agricultural Sciences,Xiqing,Tianjin 300381,China;College of life Science,Nankai University,Nankai,Tianjin 300071,China;College of Animal Science and Animal Medicine,Tianjin Agricultural University,Xiqing,Tianjin 300384,China)

机构地区：[1]天津市农业科学院畜牧兽医研究所,天津西青300381 [2]南开大学生命科学学院,天津南开300071 [3]天津农学院动物科学与动物医学学院,天津西青300384

出　　处：《中国饲料》2021年第17期10-15,共6页China Feed

基　　金：天津市农业科学院青年科研人员创新研究与实验项目(2021010);国家自然科学基金(31702095);天津市自然科学基金(18JCYBJC30200);中央引导地方科技发展专项(20ZYCGSN00030)。

摘　　要：心脏型脂肪酸结合蛋白(FABP3)是一种15 kDa的蛋白,涉及信号转导途径,参与长链脂肪酸的摄取及利用。本研究采用PCR技术扩增FABP3启动子序列,通过缺失分析构建了5个不同缺失片段荧光素酶报告基因载体,并转染奶山羊乳腺上皮细胞,通过双荧光素酶报告基因系统检测FABP3缺失片段启动子活性。结果表明:从奶山羊基因组中克隆得到2109 bp FABP3基因启动子序列(包括转录起始位点上游1985 bp),与牛(KJ649748.1)、猪(HM591296.1)和人(NG047049.1)的基因组序列同源性分别为90%、80%、75.08%。经转录因子在线软件预测分析发现,该启动子含有潜在的TATA框(TATA-box)、过氧化物酶增殖物激活受体反应元件(PPRE)、肝X受体反应元件(LXRE)、雌激素受体(ER)及两个环磷腺苷效应元件结合蛋白(CREB)的结合位点,分别位于-1632 bp和-189 bp。缺失突变研究发现,启动子片段-1801~+124活性最高,同时这一片段含有两个CREB结合位点,而当缺失至-80位点时,活性显著下降(P<0.05),且这一片段不包含CREB结合位点。表明CREB可能参与调控FABP3基因启动子的活性,为FABP3基因的转录调控研究提供理论依据。Heart fatty acid binding protein(FABP3)is a 15 kDa protein involved in signal transduction pathways and the uptake and utilization of long-chain fatty acids.In this study,the FABP3 promoter sequence was amplified by PCR,and five different length of promoter fragment luciferase reporter gene vectors were constructed through deletion analysis,and then transfected into dairy goat mammary epithelial cells.The FABP3 deletion fragment promoter activity was detected by a dual luciferase reporter gene system.The results showed that FABP3 gene promoter sequence(2109 bp including 1985 bp upstream of the transcription start site),which was cloned from the goat genome,was compatible with cattle(KJ649748.1),pig(HM591296.1)and human(NG047049.1).The genomic sequence homology was 90%,80%and 75.08%,respectively.Predictive analysis of transcription factor online software found that the promoter contains potential TATA box(TATA-box),peroxidase proliferator activated receptor response element(PPRE),liver X receptor response element(LXRE),estrogen receptor(ER)and the two cyclic adenosine phosphate response element binding proteins(CREB)elements were located at-1632 bp and-189 bp.The deletion mutation study found that the promoter fragment-1801~+124 had the highest activity,and this fragment contained two CREB binding sites.When deleted to the-80 position,the activity decreased significantly(P<0.05),and this fragment did not contain the CREB binding sites.The results indicated that CREB may be involved in regulating the activity of FABP3 gene promoter,which provides a theoretical basis for the study of FABP3 gene transcription regulation.

关 键 词：心脏型脂肪酸结合蛋白(FABP3) 启动子 基因克隆 西农萨能奶山羊 

分 类 号：S811[农业科学—畜牧学]