Tuo Decai, Shen Wentao, Yan Pu, Li Xiaoying, Zhou Peng
Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China.
Viruses. 2015 Dec 1;7(12):6241-50. doi: 10.3390/v7122935.
Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.
番木瓜叶扭曲花叶病毒（PLDMV）正成为番木瓜以及对相关病原体番木瓜环斑病毒（PRSV）具有抗性的转基因番木瓜的一大威胁。传染性病毒克隆的构建是病毒基因功能和交叉保护反向遗传学研究的关键步骤。在本研究中，一种不依赖序列和连接的克隆系统——In-Fusion（®）克隆试剂盒（美国加利福尼亚州山景城的Clontech公司），被用于构建分离株PLDMV-DF的无内含子或含内含子全长cDNA克隆，多个病毒片段和内含子片段能在单一反应中同时无痕组装到质粒载体中。PLDMV-DF含内含子的全长cDNA克隆在大肠杆菌中能稳定繁殖。含内含子的体外转录本在机械接种后经加工并剪接成具有生物活性的无内含子转录本，随后在番木瓜幼苗中引发系统感染，这些幼苗出现了与野生型病毒引起的症状相似的症状。然而，当用无内含子构建体的RNA转录本接种植物时未检测到感染性，因为病毒cDNA克隆在细菌细胞中的不稳定性导致了PLDMV-DF基因组序列的无义或缺失突变。据我们所知，这是关于构建PLDMV传染性全长cDNA克隆以及机械接种后含内含子转录本剪接的首次报道。In-Fusion克隆将构建时间从数月缩短至数天。因此，它是一种比传统的多步骤限制性内切酶介导的亚克隆程序更快、更灵活且更高效的方法。
