外源报告基因EGFP在盐藻中实现瞬时表达

探索杜氏盐藻 (Dunaliellasalina)的转基因方法和筛选方法 .利用烟草花叶病毒启动子 (CaMV35S)、衣藻叶绿体atpA启动子与来源于水母的加强型绿色荧光蛋白报告基因 (EGFP)构建表达载体pART7GFP和pUCGFP ,转化盐藻 .EGFP在CaMV 35S启动下表达出绿色荧光蛋白 ,在荧光显微镜下看到发绿色荧光的转基因盐藻 .根据荧光数目进行统计 ,转化效率高于 5 % .衣藻来源的启动子atpA在盐藻中未能启动EGFP的表达 .用直径 1μm的金粉颗粒和 0 6 μm的金粉进行基因枪法转化 ,1μm的金粉颗粒成功将外源基因导入盐藻 ,用 0 6 μm的金粉配合多种技术参数也没有将外源基因导入盐藻 .EGFP可以用作盐藻遗传转化的报告基因使单细胞真核生物盐藻可以利用流式细胞术 (FACS)等技术进行筛选 ,从而避开平板筛选转基因盐藻的限制 ,并使转基因盐藻实现无抗生素筛选成为可能

Transient Expression of Reporter Gene EGFP in Transformed Dunaliella salina

Dunaliella salina transformation and screening methods remain to be expoided. In order to know if EGFP works in D. salina , vectors pART7GFP and pUCGFP were constructed by combining EGFP with tobacco mosaic virus 35S promoter(CaMV 35S) and Chlamydomonas gene atpA promoter(atpA) respectively. Both of them were transformed into D. salina cell through microprojectile bombardment. EGFP expressed green fluorescence along with CaMV 35S, but not with atpA. According to green fluorescence, transformation efficiency reached above 5%. The function of EGFP in D. salina makes it possible to separate transformed D. salina with the aid of mammal cell sorting methods, especially FACS. Microprojectile conditions were assayed. It indicated that 1 μm gold powder was more efficient than 0 6 μm in transformaion.