Am80和TSA对鸡Stra8基因启动子活性的调控作用  

Regulatory Effects of Am80 and TSA on Chicken Stra8 Gene Promoter Activity

作　　者：刘志永[1] 左其生[1] 肖天荣 韦光辉[1] 陈庭峰 朱睿[1] 张振韬[1] 王怡临[1] 蒋舒颖 张亚妮[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《畜牧兽医学报》2015年第6期896-902,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(31272429)

摘　　要：旨在确定鸡Stra8基因启动子的主要调控区域,预测调控元件结合位点,探索用他米巴洛丁(Am80)或曲古抑菌素(TSA)诱导对Stra8启动子活性的调控作用。将鸡Stra8基因5′侧翼系列缺失片段插入到pGL3-basic载体构建重组质粒,并转染DF-1和GC-1,通过检测荧光素酶活性和预测启动子区域调控元件的结合位点,选取合适的启动子片段并构建其重组质粒Stra8-EGFP;将Am80和TSA分别按一定的浓度梯度诱导转染细胞,进行荧光素酶活性检测以筛选最佳诱导浓度;用最适剂量的Am80和TSA分别单独或联合诱导转染重组质粒Stra8-EGFP的P19,并检测各诱导组的绿色荧光表达程度。结果表明,鸡Stra8基因启动子-1 055^+54bp片段的活性较强,且含有RARα、RXRα和RARβ的结合位点;分别单独或联合使用Am80和TSA对转染的细胞进行诱导,结果显示,Am80和TSA协同作用的荧光素酶活性最高;重组质粒Stra8-EGFP转染细胞后,用Am80(10-5 mol·L-1)和TSA(10-6 mol·L-1)联合诱导可见绿色荧光强度最强。本研究成功分析了鸡Stra8基因启动子的活性和调控元件的结合位点,确定Am80和TSA共同诱导可显著提高Stra8基因启动子调控活性。The aims of this study were to identify the main regulatory regions of the chicken Stra8 gene promoter,to predict the binding sites of the transcription elements,and to investigate the regulatory effects of Am80 and TSA on the chicken Stra8 gene promoter activity.A series of promoter missing mutants were directly subcloned into pGL3-Basic vector,and the recombinant plasmids were transfected into DF-1and GC-1cells.By detecting luciferase activity and predicting binding sites of regulatory elements of promoter regulatory region,the optimal promoter fragment was selected to construct recombinant plasmid Stra8-EGFP.Am80 and TSA were applied to screen the best induced concentration,Am80 and TSA with optimal dose singly and in combination were used to induce transfected cells,and the green fluorescence expression levels in induced groups were detected.The chicken Stra8 gene promoter fragment-1 055-+54bp had stronger activity,and had the binding sites of RARα,RXRαand RARβ;Am80and TSA singly and in combination were applied to induce the transfected cells,and the luciferase activity was highest in the cells induced by Am80 and TSA in combination.After the cells were transfected by Stra8-EGFP,the expression of green fluorescence were the highest in cells induced by Am80(10-5 mol·L-1)and TSA(10-6 mol·L-1)in combination.The activity and binding sites of regulatory elements ofchicken Stra8 gene promoter were successfully analyzed,and combined actions of Am80 and TSA significantly could promote the activity of Stra8 gene promoter.

关 键 词：Stra8基因 启动子活性 调控元件 Am80 TSA 

分 类 号：S831[农业科学—畜牧学]