eGfp/Gus融合基因植物表达载体的构建和转化验证

Construction and verification of a plant vector carrying an eGfp/Gus fusion reporter gene

绿色荧光蛋白(Gfp)基因和葡萄糖醛酸糖苷酶(Gus)基因是广泛应用的2个报告基因,在定性和定量研究基因表达和启动子功能等方面各有优劣。为联合使用这2个报告基因,根据报告基因序列设计2对带有特异限制性内切酶位点的引物,PCR法分别扩增增强型Gfp(eGfp)和Gus基因,连接到植物表达载体pF-GC5941中,构建含CaMV35S启动子驱动的eGfp/Gus基因融合表达载体,命名为pFGC-DR。注射农杆菌法转化烟草叶片,以及花器官农杆菌浸泡法转化拟南芥,发现融合基因成功地在烟草叶片中瞬时表达和在拟南芥中稳定表达,表明融合报告基因在烟草和拟南芥中都能高效表达。

Green fluorescent protein（Gfp） and beta-glucuronidase（Gus） are two widely used reporter genes,which have advantages and disadvantages for qualitative analysis and quantitative determination of gene expression and promoter activity in molecular studies.A vector carrying an enhanced Gfp/Gus（eGfp/Gus） dual reporter gene was constructed to establish an efficient and convenient screening system for gene and promoter studies in plants.Primers with specific restriction endonuclease sites were designed according to sequences of the reporter genes.The eGfp and Gus genes were amplified respectively by PCR method.With corresponding endonucleases,the genes and the primary vector pFGC5941 were digested,and then these three fragments were ligated in one reaction to make a final vector.The vector,designated pFGC-DR,is characterized with a strong constitutive promoter CamV35S driving the eGfp/Gus fusion gene.Transient expression in tobacco leaves and transgenic expression studies in Arabidopsis showed that this fusion reporter protein retains functional activity for both eGfp and Gus.These results demonstrated the utility of the eGfp/Gus dual reporter system for studying of plant promoters.