尼罗罗非鱼β-actin基因启动子的分离及其在真核细胞中的活性验证  

Isolation of β-actin gene promoter of nile tilapia and its activity identification in eukaryotic cells

作　　者：邹芝英[1] 王茂元[1] 李大宇[1] 杨弘[1] 

机构地区：[1]中国水产科学研究院淡水渔业研究中心农业部淡水鱼类遗传育种和养殖生物学重点开放实验室,江苏无锡214081

出　　处：《淡水渔业》2011年第1期78-82,共5页Freshwater Fisheries

基　　金：国家科技支撑计划专题(2006BAD01A1202);2007年公益性行业(农业)科研专项(3-49);现代农业产业技术体系建设专项资金(nycytx-48);国家重点基础研究发展计划973计划项目(2004CB117401)

摘　　要：采用高保真PCR方法从尼罗罗非鱼(Oreochromis niloticus)基因组DNA中分离出β-actin基因启动子序列,将β-actin基因启动子插入pN1-EGFP构建成真核细胞表达载体pEGFP-β-actin,并通过脂质体转染法将重组载体导入人胚肾上皮细胞HEK 293T,荧光显微镜下观察外源基因EGFP在细胞中的表达情况。结果显示:β-actin基因启动子调控区具有典型的启动子特征,含有TATA Box、CArG和CCAAT Box 3个重要的顺式作用元件。荧光显微镜下观察到50%～60%的HEK 293T细胞中发出绿色荧光,显示尼罗罗非鱼β-actin基因启动子能够驱动EGFP的转录和表达,具有较好的活性,为构建"全鱼"转基因罗非鱼的研究奠定基础。Using high-fidelity PCR method,1675 bp DNA fragment of β-actin gene promoter was cloned from the genomic DNA of Nile tilapia（Oreochromis niloticus）.One the other hand,with the EGFP reporter gene,the plasmid pN1-EGFP was constructed without promoter.The eukaryotic expression vector pEGFP-β-actin was recombined with insertion of the gene promoter.By lipofetamine-mediated transfection,the recombined vector was induced into HEK 293T cell line.The expression level of the EGFP protein was detected under the fluorescence microscopy.Sequence analysis showed that the regulatory region of β-actin gene promoter had the typical characteristics,consisting of 3 important homeopathic components TATA Box,CArG and CCAAT Box.Under the microcopy,50%~60% of the HEK 293T cells detected were in the green fluorescence.It was indicated that the promoter could drive the translation and expression of EGFP,which confirmed that the cloned Nile tilapia β-actin gene promoter had higher activity.So the present study lay the foundation for the construction of ＇all-fish＇ transgenic tilapia.Meanwhile,it explored the establishment of the rapid detection of the promoter activity of tilapia in eukaryotic cells.

关 键 词：尼罗罗非鱼(Oreochromis niloticus) Β-肌动蛋白 启动子 绿色荧光蛋白 转染 

分 类 号：Q78[生物学—分子生物学]