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

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...

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

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.