摘要
【目的】固醇调节元件结合蛋白1(SREBP1)作为核转录因子,对于细胞脂肪合成酶基因的表达发挥着重要的调控作用。论文旨在奶牛乳腺上皮细胞中研究SREBP1对于SCD1基因启动子的转录调控作用,为进一步明确SREBP1对于靶基因的转录调控机制提供理论基础。【方法】以荷斯坦奶牛乳腺组织的c DNA为模板,采用分段克隆的方法获得SREBP1基因的编码序列,通过重组酶与pc DNA3.1载体进行重组环化构建pc DNA3.1-SREBP1表达载体,将构建的载体测序验证后提取质粒,转染奶牛乳腺上皮细胞。以EIF3K基因为内参基因,采用荧光定量PCR检测SREBP1基因m RNA的表达差异;采用免疫荧光的方法对SREBP1进行标记,以DAPI复染细胞核,激光共聚焦观察SREBP1蛋白的亚细胞定位;转染含有不同调控元件的SCD基因启动子,同时转染1.0μg pc DNA3.1-SREBP1作为处理,荧光素酶报告基因系统分析启动子活性;分别转染0.25、0.5和1μg的pc DNA3.1-SREBP1载体,分析p GL3-SCD 2和p GL3-SCD3启动子活性与SREBP1之间的量效关系。【结果】分段克隆得到的PCR产物分别为1 170、1 116、363和900 bp的片段,经过与pc DNA3.1载体重组后获得pc DNA3.1-SREBP1表达载体,经酶切和测序验证,发现除1个无义突变外,与标准序列完全相同,整个序列长度达到3 510bp;将pc DNA3.1-SREBP1载体转染乳腺上皮细胞后,Real-time PCR检测发现与转染空载体的对照组相比,SREBP1基因的m RNA表达倍数增强130.4倍(P<0.001);激光共聚焦观察发现,DAPI染色的细胞核呈蓝色,免疫荧光标记的SREBP1呈绿色,二者融合后呈现青色,共定位在乳腺上皮细胞核中;启动子活性检测发现,与p GL3-SCD1、p GL3SCD 2相比,SREBP1处理能够极显著增加p GL3-SCD3、p GL3-CD4启动子的活性(P<0.001),分别比对照组提高了1.0倍和0.7倍,进一步分析发现,在用0.25—1μg的pc DNA3.1-SREBP1处理后,与p GL3-SCD2的启动子活性持续下降相比,p GL3-SCD3的启动子活性从59.81上升到108.43(P<0.001),二者存在剂量效应关系,结合SCD2和SCD 3启动子上主要的结构差异SRE元件(5′-AGCAGATTGCG-3′),推测此序列可能是SREBP1调控SCD基因启动子转录的结合序列。【结论】克隆构建奶牛SREBP1基因表达载体,亚细胞定位SREBP1蛋白主要在乳腺上皮细胞核中,SREBP1可以与SRE调控元件结合促进SCD1基因启动子的转录。
【Objective】As a member of nuclear transcription factor,sterol regulatory element binding protein1（SREBP1）plays a significant role in the expression of the lipogenic gene.The objective of this study is to investigate the effect of SREBP1 on the regulation of transcription of SCD1 gene in the mammary epithelial cells,which will provide a fundamental basis for the transcription regulation mechanism of SREBP1 to target gene.【Method】 The coding sequence（CDS） of SREBP1 gene was cloned using the method of subsection cloning with the cDNA of Holstein mammary tissues as the template.The expression vector pc DNA3.1-SREBP1 was recombined and constructed by recombining vector pc DNA3.1.Plasmids were extracted from the constructed vector after being verified by sequencing,and transfected to mammary epithelial cells.With gene EIF3 K as the reference gene,the expressional difference of m RNA in gene SREBP1 was detected with Real-time PCR.SREBP1 was marked with the immunofluorescence method.The cell nucleus was restained with DAPI,and the subcellular localization of protein SREBP1 was observed with laser con-focal microscopy.The SCD1 gene promoters with different regulatory elements and 1.0 μg pc DNA3.1-SREBP1 were restained as treatments,and the promoter activity was analyzed systematically with luciferase reporter gene.Vectors pc DNA3.1-SREBP1 of 0.25 μg,0.5 μg and 1 μg were transfected respectively.The dose-effect relationship between the promoter activity of p GL3-SCD2,p GL3-SCD3 and protein SREBP1 was analyzed.【Result】 The PCR products cloned in subsection were fragments 1170 bp,1116 bp,363 bp and 900 bp respectively.The expression vector pc DNA3.1-SREBP1 was obtained by reconstructing the fragments and vector pc DNA3.1.Confirmed by digestion and sequence,the expression vector,except one nonsense mutation,was exactly the same with the standard sequence.The whole sequence length reached 3510 bp.After vector pc DNA3.1-SREBP1 was transfected into mammary epithelial cells,compared with the control group of the empty transfected vector,the expression m RNA of gene SREBP1 increased by 130.4 times（P0.001）by the real-time PCR detection.As shown in the observation of the laser con-focal microscopy,the cell nucleus dyed with DAPI was blue,and SREBP1 with immunofluorescent labeling was green,both of which were cyan after being merged and were localized in the mammary epithelial cell nucleus.The results of the detection of the promoter activity showed that,compared with those of p GL3-SCD1 and p GL3 SCD 2,the treatment with SREBP1 increased the promoter activity of p GL3-SCD3 and p GL3-CD4 significantly（P0.001）,and their promoter activity increased by 1.0 and 0.7 times.The further study showed that,after treatment with 0.25-1μg pc DNA3.1-SREBP1,compared with the continuous decrease of the promoter activity of p GL3-SCD2,the promoter activity of p GL3-SCD3 increased from 59.81 to 108.43（P0.001）.There is a dose-effect relationship between them.Combined with the element SRE（5′-AGCAGATTGCG-3′）,the architectural difference between promoter SCD2 and promoter SCD 3,it could be speculated that the sequence was the incorporating sequence in which SREBP1 regulated the transcription of gene promoter SCD1.【Conclusion】 It was verified that the gene expression vector SREBP1 was constructed by cloning,the subcellular localization of protein SREBP1 was in the mammary epithelial cell nucleus,and SREBP1 promoted the regulation of the transcription of SCD1 promoter together with regulatory element SRE.
出处
《中国农业科学》
CAS
CSCD
北大核心
2016年第24期4797-4805,共9页
Scientia Agricultura Sinica
基金
国家"973"项目(2011CB100802)
国家自然科学基金(31072009)
国家转基因重大专项(2014ZX0801015B)
河南省高等学校重点科研项目(15A230020)
河南省基础与前沿项目(162300410258)
