基于黄瓜花叶病毒基因组RNA2的外源基因表达载体研究

Study on Exogenous Gene Expression Vector Based on Cucumber Mosaic Virus Genome RNA2 in Nicotiana Benthamiana

为了确定黄瓜花叶病毒(Cucumber mosaic virus,CMV)基因组RNA2能否作为构建表达外源蛋白的载体,实验构建CMV的Fny株系(CMV-Fny)RNA2中2b基因缺失载体pCB301-F209Δ2b和携带绿色荧光蛋白(green fluorescent protein,GFP)基因载体pCB301-F209Δ2b-gfp,并分别将含有质粒CMV-FnyΔ2b和CMVFnyΔ2b-gfp农杆菌混和物浸润接种寄主植物。结果表明:CMV-FnyΔ2b在本氏烟产生轻微花叶症状并且病毒基因组含量降低;在CMV-FnyΔ2b-gfp侵染植株叶片中有绿色荧光的产生;番茄丛矮病毒沉默抑制子p19加入有助于CMV-FnyΔ2b表达GFP;在RDR6含量减少的本氏烟RDR6i中,与对照接种植株相比,CMV-FnyΔ2b-gfp所产生的GFP增加。研究获得基于CMV基因组RNA2所构建的外源蛋白表达载体CMV-CMV-FnyΔ2b,并发现加入其它病毒基因沉默抑制子有利于GFP的表达。

In order to verify whether Cucumber m osaic virus （CMY） genome RNA2 could serve as the vector to express exogenous protein, 2b gene deletion vector pCB301-F209△2b and pCB301-F209△2b- gfp carrying green fluorescent protein （GFP） in CMV Fny strain RNA2 were constructed in the experiments. The agrobacterium mixtures containing plasmids CMV-FnyA2b and CMY-Fny△2b gfp was respectively inoculated into the host plant. The results indicate that CMV-FnyA2b generated slight mosaic symptom in Nicotiana benthamiana and the level of viral genome reduced. Green fluorescence emerged in the plant leaves infected by CMV-Fny△2 b- gfp . The presence of gene silencing suppressor protein p 19 from Tomato bush stunt virus in the inoculum of CMV-Fny△2b- gfp contributed to GFP expression by CMV-Fny△2b. In N. benthamiana RDR6i with reduced RDR6 accumulation, GFP generated by CMY-Fny△2 b gfp increased, compared with the control plant. In this study, exogenous protein expression vector CMV- FnyA2b based on CMV genome RNA2 was gained, and it was found that the addition of other virus gene silencing suppressor contributed to GFP expression.