State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China.
Yunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming 650021, China.
Viruses. 2017 Nov 7;9(11):332. doi: 10.3390/v9110332.
The availability of infectious full-length clone is indispensable for reverse genetics studies of virus biology, pathology and construction of viral vectors. However, for RNA viruses with large genome sizes or those exhibiting inherent cloning difficulties, procedure to generate biologically active circular DNA (cDNA) clones can be time-consuming or technically challenging. Here we have constructed a yeast-- shuttle vector that enables highly efficient homologous recombination in yeast for assembly of compatible plant virus clones. Using this vector, we show that infectious cDNA clones of a plant negative-stranded RNA virus, sonchus yellow net rhabdovirus, can be rapidly assembled. In addition, one-step assembly of infectious clones of potato virus Y in yeast, either with or without intron, was readily achieved from as many as eight overlapping DNA fragments. More importantly, the recovered yeast plasmids can be transformed directly into for inoculation, thereby obviating the cloning steps and associated toxicity issues. This method is rapid, highly efficient and cost-effective and should be readily applicable to a broad range of plant viruses.
传染性全长克隆的可用性对于病毒生物学、病理学和病毒载体的构建的反向遗传学研究是必不可少的。然而，对于基因组较大或具有固有克隆困难的 RNA 病毒，生成具有生物活性的环状 DNA(cDNA)克隆的过程可能既耗时又具有技术挑战性。在这里，我们构建了一种酵母穿梭载体，该载体能够在酵母中进行高效的同源重组，从而组装兼容的植物病毒克隆。使用该载体，我们表明可以快速组装植物负链 RNA 病毒——黄花叶病毒的传染性 cDNA 克隆。此外，来自多达 8 个重叠 DNA 片段的马铃薯 Y 病毒的有或没有内含子的感染性克隆可以在酵母中一步组装。更重要的是，回收的酵母质粒可以直接转化为侵染，从而省去了克隆步骤和相关的毒性问题。该方法快速、高效且具有成本效益，应该易于应用于广泛的植物病毒。
