一种根癌农杆菌介导的GFP亚细胞定位方法的优化

An Optimized Method of Agrobacterium tumefaciens-mediated GFP Subcelluar Localization

利用根癌农杆菌介导的方法,采用CaMV 35S强启动子启动的含有GFP报告基因的双元表达载体pCAMBIA1301-35S-GFP-AZI1转化烟草表皮细胞。在激光共聚焦显微镜下进行观察,有绿色荧光产生。本文优化了GFP亚细胞定位的方法,即烟草为本氏烟草2～3周龄叶片,菌液OD600值为0.8,共培养时间为32 h,在此条件下能很好地进行GFP的亚细胞定位,为进一步研究新基因的亚细胞定位和瞬时表达奠定了基础。

By using the Agrobacterium tumefaciens-mediated method, the binary expression vector, pCAMBI- A1301-35S-AZI1-GFP was used to study the subcellular location, the vector with GFP report gene that is strongly activated by the strong CaMV 35S promoter to transform tobacco lower epidermal surface of tobacco leaves. The green fluorescence of GFP was observed by using laser confocal microscope. In this paper, the method of GFP subcelluar location was optimized, such as 2-3 week-old leaves of Bens tobacco, bacterium solution OD600=0.8, 32 h dark incubation. The appropriate result ofGFP subcelluar location can be obtained under the above conditions. The technologies would be widely employed to location of a new gene and expression in further research.