农杆菌介导的烟草瞬时表达试验条件优化

Transient Expression Conditions of Tobacco Transformation Mediated by Agrobacterium

以整株烟草为试材,用转入植物表达载体p CAMBIA1301的根癌农杆菌EHA105进行遗传转化。通过分析不同菌液浓度、乙酰丁香酮浓度、Tween20浓度、蔗糖浓度、不同侵染时间及有无激素对GUS基因表达的影响,研究其瞬时表达的最佳条件。结果显示:用OD600值为0.2的农杆菌侵染3 h,在1/2 MS培养基中添加120μmol/L的乙酰丁香酮,1.5 mg/L的KT,0.5 mg/L的NAA,3%的蔗糖,共培养3 d,能够得到高效的GUS基因瞬时表达。

An exp erime nt was conducted to investigate the best conditions of transient expression of the whole tobacco plant(Nicotianatabacum L.) that transformed with the Agrobacterium tumefaciens EHAl05 containing the plant expression vector p CAMBIA1301 by Agrobacterium mediated approach. Effects of the concentrations of A. tumefaciens, acetosyringone, Tween20, and sucrose, infection time, and hormone on the transient expression of GUS gene were analyzed to determine the optimal transformation conditions. A higher transient expression level of GUS gene could be obtained as OD600 value of A. tumefaciens for infiltration 0.2, infiltrating for 3 h,adding 120 μmol/L acetosyringone, 1.5 mg/L KT, 0.5 mg/L NAA, and 3% sucrose to co-culturemedium, and coculture for 3 days.