农杆菌介导的油菜子叶瞬时表达

An agrobacterium-mediated transit transformation system in Brassica napus cotyledon

实验以甘蓝型油菜宁油16为材料,绿色荧光蛋白(GFP)为报告基因,建立了农杆菌介导的油菜子叶瞬时表达系统。我们构建了GFP表达载体pB2GW7.0-gfp,并成功转化农杆菌。实验中通过添加P19来提高GFP在油菜子叶中的瞬时表达量。并提取了注射有P19和pB2GW7.0-gfp农杆菌混合液的油菜子叶RNA,经RT-PCR鉴定,发现在4～8 d GFP均能表达。激光共聚焦显微镜分析表明农杆菌介导的油菜子叶瞬时表达系统能够转化油菜子叶的表皮细胞和保卫细胞。甘蓝型油菜子叶瞬时表达方法简便、快速、可靠,从种子播种到获得荧光蛋白表达,全过程只需要20 d。表明在研究油菜基因的表达和功能方面有潜在的应用前景。

A method for transient expression based on Agrobacterium infiltration into cotyledons of Brassica napus L.cv Ningyou 16 was developed with the Green Florescent Protein（GFP） as a marker gene.GFP expression vector pB2GW7.0-gfp was constructed and transferred into agrobacterium.The addition of P19 protein improved the transient expression level of GFP in cotyledon.Reverse transcription assay showed that GFP was expressed after 4-8 days of agrobacterium infiltration.Confocal microscopical analysis revealed that epidermal cells and guard cells could be transformed by agrobacterium-based transformation method.The entire process only took 20 days from sowing seed to protein analysis.Therefore,this new method is simple and rapid.It has a potential application in dissection genes expression and function in Brassica napus.