7种RNA沉默抑制子对植物病毒载体表达系统表达水平的影响  

Effects of seven RNA silencing suppressors on heterologous expression of green fluorescence protein expression mediated by a plant virus-based system in Nicotiana benthamiana

作　　者：王盛[1,2] 董洁[1] 曹慜[1] 穆红珍[1] 丁国平[1] 张虹[1] 

机构地区：[1]宁夏大学生命科学学院 [2]西部特色生物资源保护与利用教育部重点实验室,宁夏银川750021

出　　处：《南方医科大学学报》2012年第11期1536-1542,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(31060023;31160032);教育部科学技术研究重点项目(209135)~~

摘　　要：目的探索不同植物病毒RNA沉默抑制子对植物病毒载体表达系统重组蛋白表达水平的作用,为合理高效地利用这一新型的外源基因表达平台奠定基础。方法构建了7种不同的RNA沉默抑制子瞬时表达载体,以农杆菌渗滤法,与马铃薯X病毒表达载体PVXdt-GFP共侵染寄主植物本明烟,通过对报告基因绿色荧光蛋白(GFP)的荧光观察,并以Western blotting、ELISA和RT-qPCR等测定GFP在烟草中的表达情况,分析不同RNA沉默抑制子对植物中外源基因表达水平的作用和特点。结果 7种病毒RNA沉默抑制子对外源基因GFP在烟草中表达水平的作用效果和持续时间存在差异,其中,番茄丛矮病毒的P19蛋白的增效作用最好,作用时间也最长;非洲木薯花叶病毒的AC2蛋白和水稻黄斑驳病毒的P1蛋白无明显的增效作用。结论 RNA沉默抑制子可以通过抑制病毒诱导的RNA沉默来提高外源基因在植物中的表达水平和表达持续时间,但是不同的植物病毒载体表达系统需要通过筛选获得其最佳的共表达沉默抑制子"伴侣"。Objective To test the effects of 7 virus-encoded RNA silencing suppressors （RSSs） for enhancement of a plant virus-based vector system-mediated heterologous expression of green fluorescence protein （GFP） in Nicotiana benthamiana. Methods Seven transient expression vectors for the 7 RSSs were constructed and co-inoculated on the leaves of Nicotiana benthamiana with PVXdt-GFP vector, a novel Potato virus X-based plant expression vector, through agroinfiltrafion. The protein and mRNA expression levels of the reporter gene GFP in the co-inoculated Nicotiana leaves were examined by Western blotting, ELISA and RT-qPCR to assess the effect of the RSSs for GFP expression enhancement. Results The 7 RSSs differed in the degree and duration of enhancement of heterologous GFP expression, and the p19 protein of Tomato bushy stunt virus （TBSV） induced the highest expression of GFP. African cassava mosaic virus AC2 protein and Rice yellow mettle virus P1 protein produced no obvious enhancement GFP expression. Conclusion Transient co-expression of RSSs suppresses host silencing response to allow high-level and long-term expression of heterologous genes in plant, but the optimal RSS has to be identified for each plant virus-based expression vector system.

关 键 词：RNA沉默抑制子 马铃薯X病毒 共表达 绿色荧光蛋白 

分 类 号：Q943.2[生物学—植物学]