禽U6启动子的克隆、序列分析及其驱动的siRNA表达  

Cloning,Sequence Analysis and Transcription Activity of a Chicken U6 Promoter

作　　者：王永娟[1,2] 沈鹏鹏[1] 张鑫宇[1] 夏晓莉[1] 孙怀昌[1] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏畜牧兽医职业技术学院,江苏泰州225300

出　　处：《中国农业科学》2009年第8期3003-3008,共6页Scientia Agricultura Sinica

基　　金：国家自然科学基金资助项目(30460090)

摘　　要：【目的】分离用于禽源细胞中表达短干扰RNA(short interference RNA,siRNA)的禽U6启动子。【方法】以鸡基因组为模板进行PCR扩增,将扩增产物进行序列测定和生物信息学分析,并将其插入报告基因载体pEGFP-N1中,获得siRNA表达载体pGFP-U6,将由软件预测针对GFP基因的siRNA所对应的shRNA(short hairpin RNA)插入其中,获得pGFP-U6-shRNA;以含人H1启动子的siRNA表达载体pGFP-H1-shRNA为对照,分别转染COS-1和DF-1细胞,用荧光显微镜和流式细胞仪测定转染细胞培养中GFP阳性细胞数和荧光总量。【结果】克隆的U6启动子位于鸡28号染色体,与发表的鸡U6-3启动子同源性为97.2%,含多个Oct-1序列,不含CACCC框、SPH和PSE等聚合酶Ⅲ启动子序列,TA框不典型;在pGFP-U6-shRNA转染的哺乳动物源COS-1细胞培养中,GFP阳性细胞数和荧光总量均有所下降,但不如pGFP-H1-shRNA转染细胞显著;在pGFP-U6-shRNA转染的禽源DF-1细胞培养中,不仅GFP阳性细胞数和荧光总量显著下降,而且基因沉默效果显著优于pGFP-H1-shRNA转染细胞。【结论】成功克隆的禽U6启动子不仅能有效转录siRNA,而且转录活性具有相对的细胞种属特异性。[ Objective ] To cloning avian U6 promoter for construction of short interference RNA （siRNA） expression vectors in avian cells. [Method] A putative chicken U6 promoter from chicken genomic DNA was amplified by PCR. After sequencing and bioinformatics analysis, the putative promoter was inserted into the reporter plasmid pEGFP-N1 together with GFP-specific short hairpin RNA （shRNA） and resulted in a siRNA expression vector pGFP-U6-shRNA. Then, using human H1 promoter-driven siRNA expression vector pGFP-H 1-shRNA as the control, the simian-originated COS-1 cells or chicken embryonic fibroblast （DF-1） cells were cotransfected with the siRNA expression vector pGFP-U6-shRNA, and the GFP-positive cell numbers and total fluorescence were detected by fluorescence microscopy and flow cytometry. [ Result ] The PCR product was 560bp long and located on chicken chromosome 28 with a percentage identity of 97.2% to the published chicken U6-3 promoter. Bioinformatics analysis showed that the putative chicken U6 promoter contained several Oct-1 motifs and a weak TATA box without other Pol Ⅲ promoter elements. In COS-1 cells, transfection with pGFP-U6-siRNA resulted in significant decreases in both the number of GFP-positive cells and total fluorescence, but significance was lower than that of transfection with pEGFP-H1-shRNA. While in DF-1 cells, transfection with pGFP-U6-shRNA resulted in significantly higher gene silencing effect than cotransfection with pEGFP-HI-shRNA. [ Conclusion] These data suggest that a chicken U6 promoter was successfully cloned, which can efficiently transcribe gene-specific siRNA expression in avian cells.

关 键 词：禽U6启动子 克隆 序列分析 转录活性 

分 类 号：Q78[生物学—分子生物学]