Choi Sun Hee, Ryu Ki Hyun
Plant Virus GenBank, Division of Environmental and Life Sciences, Seoul Women's University, Seoul, 139-774, Republic of Korea.
J Microbiol. 2008 Oct;46(5):502-7. doi: 10.1007/s12275-008-0078-2. Epub 2008 Oct 31.
Full-length cDNAs of two different strains of Potato virus X (PVX-Kr and PVX-Mo) have been directly amplified by long template reverse transcription polymerase chain reaction (RT-PCR) using the 5'-end primer containing a SP6 or T7 RNA promoter sequence and the virus-specific 3'-end primer, and then constructed in plasmid vectors. Capped in vitro transcripts from cloned full-length cDNAs as well as those RT-PCR amplicons proved to be infectious systemically on tobacco plants. Symptom expression on tobacco plants from PVX-Mo transcripts was faster and severer than that from PVX-Kr. In replication stability test of transcripts derived from PVX clones, progeny viruses showed stable replication according to sequencing through passages. This highly infectious transcript system from the full-length cDNA clones for PVX can be useful for recombinant molecules for functional analysis of viral proteins in plant-virus interaction study as well as for expression of foreign protein in planta.
利用含有SP6或T7 RNA启动子序列的5'端引物和病毒特异性3'端引物,通过长模板逆转录聚合酶链反应(RT-PCR)直接扩增了两种不同马铃薯X病毒株(PVX-Kr和PVX-Mo)的全长cDNA,然后构建到质粒载体中。克隆的全长cDNA以及那些RT-PCR扩增产物的加帽体外转录本在烟草植株上被证明具有系统感染性。PVX-Mo转录本在烟草植株上引起的症状表达比PVX-Kr的更快、更严重。在PVX克隆转录本的复制稳定性测试中,子代病毒通过传代测序显示出稳定的复制。这种来自PVX全长cDNA克隆的高感染性转录本系统可用于植物-病毒相互作用研究中病毒蛋白功能分析的重组分子,以及用于在植物中表达外源蛋白。