Hasiów-Jaroszewska Beata, Borodynko Natasza, Pospieszny Henryk
Department of Virology and Bacteriology, Institute of Plant Protection, National Research Institute, ul. Wegorka 20, 60-318 Poznan, Poland.
Arch Virol. 2009;154(5):853-6. doi: 10.1007/s00705-009-0368-y. Epub 2009 Mar 31.
For the first time, a full-length cDNA clone of the RNA genome of pepino mosaic virus (PepMV) was constructed. RNA was extracted from purified virions of isolate PepMV-Pa and used for cDNA synthesis. The full-length cDNA was produced as one 6.4-kb fragment representing the entire PepMV genome. This fragment was ligated into the pCR-XL-TOPO vector downstream of T7 RNA polymerase promoter, which was included in the 5' primer sequence used for RT-PCR. The PepMV-Pa RNA transcripts obtained were infectious in different host plants, causing symptoms indistinguishable from those of the wild-type isolate. The presence and authenticity of the progeny virus were verified by ELISA, RT-PCR and nucleotide sequencing.
首次构建了番木瓜斑驳病毒（PepMV）RNA基因组的全长cDNA克隆。从分离株PepMV-Pa的纯化病毒粒子中提取RNA，并用于cDNA合成。全长cDNA作为一个代表整个PepMV基因组的6.4 kb片段产生。该片段被连接到T7 RNA聚合酶启动子下游的pCR-XL-TOPO载体中，T7 RNA聚合酶启动子包含在用于RT-PCR的5'引物序列中。获得的PepMV-Pa RNA转录本在不同宿主植物中具有感染性，引起的症状与野生型分离株无法区分。通过ELISA、RT-PCR和核苷酸测序验证了子代病毒的存在和真实性。
