基于烟草脆裂病毒构建同时表达2种外源蛋白的载体及其应用

目的 构建在整个寄主植物中能同时表达两个非融合蛋白的病毒载体。方法 以烟草脆裂病毒（tobacco rattle virus,TRV）基因组RNA2的农杆菌侵染性克隆pYL156为材料,缺失TRV RNA2的2b基因5"端279 bp并将其起始密码子ATG改变为AGG、同时引入豌豆早枯病毒（pea early-browning virus,PEBV）外壳蛋白（coat protein,cp）基因启动子,获得pTRV2e²载体;在pTRV2e²载体的2b和PEBV的cp启动子下游插入不同的外源基因,测定病毒TRVe²表达外源蛋白的能力、携带外源基因后重组病毒的稳定性以及分析蛋白质的生物学功能。结果 病毒TRVe²能快速同时表达2个非融合的外源蛋白,且至少能表达70 ku外源蛋白,且该病毒携带~2.0 kb外源基因能稳定存活于寄主植物中;病毒TRVe²可用于分析蛋白质的生物学功能以及2个蛋白质间的相互作用。结论 本文构建的重组病毒TRVe²为快速有效地表达2个外源蛋白以及分析2个蛋白质间的相互作用提供技术工具。

Construction of Dual Expression Vector Based on Tobacco Rattle Virus and Its Application for Analysis of Protein Function in Nicotiana benthamiana

Objective In order to construct a viral vector that can simultaneously express two non-fusion proteins in the whole host plants.Methods Agrobacterium infectious clone pYL156 containing tobacco rattle virus (TRV) genomic RNA2 was used to construct dual expression vector pTRV2e² by deleting 279 bp of 5" end of 2b gene, changing initiation codon of 2b gene ATG to AGG, and introducing the subgenomic promoter of pea early-browning virus (PEBV) coat protein (cp) gene. Different exogenous genes were cloned into the downstream of 2b and PEBV cp subgenomic promoters to measure the ability of virus TRVe² to express two foreign proteins, assess the stability of reconstructed TRVe² and analyze the function of proteins in the seedlings of Nicotiana benthamiana.Results TRVe² could simultaneously and rapidly produce two non-fused target proteins and express at least a 70 ku foreign protein in the whole host plants; TRVe² harboring 2.0 kb exogenous gene could stably exist in N. benthamina plants and could be served as technical means for analyzing the biological function of the proteins and the interaction between two proteins.Conclusion Recombinant virus TRVe² constructed in this study provide a toolbox for fast and efficient production of double foreign proteins and for analysis of the interaction between two proteins in the host plants.