绿色荧光蛋白标记检测鸦胆子素D抗TMV活性

Detection of Anti-TMV Activity of Bruceine D by Green Fluorescent Protein Labeling

以绿色荧光蛋白(green fluorescent protein,GFP)为报告基因,将含TMV表达载体的质粒p35S-30B:GFP转化农杆菌EHA 105,通过渗透法把经MMA诱导后的农杆菌悬浮液注射到本氏烟叶片内,测定了鸦胆子素D(Bruceine D)对烟草植株内TMV的增殖和运动的抑制作用;通过PEG介导法把p35S-30B:GFP转化到本氏烟叶肉细胞原生质体内,测定了Bruceine D对烟草原生质体中TMV增殖的抑制效果。结果表明,在10μg/mL浓度下,Bruceine D不仅可抑制烟草叶肉细胞原生质体中TMV的增殖,还可以抑制烟草接种叶中TMV向茎部及植株上部叶片移动,且对寄主植物不造成明显的毒害。

To investigate the effects of Bruceine D on multiplication and movement of TMV in Nicotiana benthamiana, the green fluorescent protein （GFP） gene was used as a reporter gene, and agrobacterium strain EHA 105 harbouring TMV based expression vector p35S-30B：GFP was infiltrated into Nicotiaaa benthamiana leaves after induction by MMA. For assessment of Bruceine D inhibitting replication of TMV in tobacco protoplasts, p35S-30B：GFP after PEG-mediated was delivered into protoplasts derived from Nicotiana benthamiana leaves. The results showed that Bruceine D could not only inhibit the multiplication of TMV in the protoplasts, but also restrict systemic movement of TMV in tobacco at 10 μg/mL, while no significant negative effects on the normal physiology of the tested plants were observed.