TBSV病毒瞬时表达载体构建及其表达

Construction of TBSV RNA-Based Transient Expression Vectors and Its Expression in Tobacco Plants

为了深入理解植物RNA病毒载体表达系统,初步建立了利用TBSV病毒表达载体在寄主植物烟草中表达外源蛋白的技术体系.以番茄丛矮病毒（Tomato bushy stunt virus,TBSV）为材料,构建了含报告基因sGFP的WY1,WY2等2个重组病毒表达载体,并将载体导入农杆菌GV3101用于侵染烟草植株叶片;利用农杆菌渗滤接种技术,使构建的载体在烟草本氏烟（Nicotiana benthamiana）中进行瞬时表达.结果表明,接种WY1的烟草,于接种后第2 d就可以检测到荧光信号,10~12 d时表达量达到最高.纯化后的sGFP与标准GFP相对分子质量相同且其荧光光谱也与文献报道的一致.对于感兴趣基因（Gene of interested,GOI）的表达和研究提供了一种新的途径.

In order to better understand the utility of plant virus RNA-based expression system,we established a TBSV RNA-based expression system for transient expression of proteins in tobacco.Constructs were generated with full-length Tomato bushy stunt virus（TBSV） cDNA and two TBSV-derived expression vectors（WY1 and WY2） were constructed.Those vectors were delivered into Tobacco plants（Nicotiana benthamiana）by means of an agrobacterium-mediated transient transformation assay.The results show that the fluorescent signal of GFP can be detected in agroinfiltrated leaves（agroinfiltrated by replicon WY1） by two days post infiltration（DPI） and maximal accumulation achieved by 10—12 DPI.Purified GFP proteins are consistent with previous reports in molecular weight and fluorescence spectrum characteristics.This new transient expression vector will be a useful tool for expressing recombinant proteins in plants for either research or production purposes.