植物RNA病毒载体表达系统是一种极具潜力的新型外源基因表达平台。当前植物RNA病毒载体均使用Ⅱ型启动子，与Ⅱ型启动子伴生的转录后加工（加帽、加尾和内含子剪接等）会影响重组病毒基因组RNA的完整性，导致载体表达效率下降、外源基因丢失和产物不均一等问题。Ⅰ型启动子转录活性高、转录产物无需类似加工且具有种属间特异性，应用于病毒表达载体即利于保持转录后重组病毒基因组的完整性，也利于提高系统的生物安全性。鉴于此，本项目在克隆本氏烟Ⅰ型启动子的基础上，以番茄丛矮病毒为实验对象，以GFP为报道基因，构建本氏烟Ⅰ型启动子介导转录的病毒表达载体。通过相关研究，探明启动子中相关顺式调控元件及其功能；明确转录后RNA加工对病毒表达系统的影响；建立Ⅰ型启动子介导转录的病毒表达系统并评价系统在提高生物安全性和实用性上的潜力。研究对深入理解植物病毒表达系统和指导新类型病毒表达载体的构建方面均具有理论意义和借鉴价值。

As a novel expression system, plant virus RNA-based vectors allow for the rapid, high level, transient expression of proteins in whole plants. Many different types of plant viruses have been converted into vectors for the production of recombinant proteins or peptides and they all used the type II promoters. However, under natural conditions, plant RNA viruses replicate in the cytoplasm and are not adapted to the nuclear splicing machinery, which recognizes and removes cryptic introns from viral RNA. As a result, pre-mRNA transcripts made in the nucleus from viral constructs may not be recognized and processed properly and the formation of active replicons from the primary nuclear transcript is inefficient. RNA Pol I is a cellular enzyme that is abundantly expressed in cells and transcribes rRNA precursor lacking a 5′ cap, a 3′ poly (A) tail and introns. Thus, viral RNA synthesized in cells transfected with Pol I-driven plasmids containing viral genomic cDNA has precise sequences. This may increase the expression level and biological safety of the plant RNA-based expression system. In addition to, Nicotiana benthaminana is a kind of model organism which widely used in plant bioreactor and plant pathology. For these reasons, we plan to clone the Nicotiana benthaminana Pol I promoter sequence and analyses its transcription initiation site. And then the TBSV will be used to construct the infectious cDNA vectors under control of the Nicotiana benthaminana rRNA promoter. Based on those constructed vectors, we will verify the cis-elements and its function in the Nicotiana benthaminana Pol I promoter sequence and exploit if Pol I-directed transcription require nuclear splicing. After that, a RNA polymerase I-mediated plant virus RNA-based expression system will be established. The biological safety and utilities of this Pol I-driven plant RNA-based expression system also be evaluated simultaneously. The related results from this study will be beneficial for the further research of both Pol I promoter and Pol I-based plant virus expression vectors. Moreover, this new transient expression system will be a useful tool for expressing recombinant proteins in plants for either research or production purposes.