Phytopathology. 1999 Dec;89(12):1182-5. doi: 10.1094/PHYTO.1999.89.12.1182.
ABSTRACT Infectious RNA of wheat streak mosaic virus (WSMV) has been produced using a full-length cDNA clone as a template for in vitro transcription with SP6 RNA polymerase. Infectivity was dependent on the use of template plasmid DNA that had not undergone spontaneous rearrangement during amplification in Escherichia coli. The presence of WSMV in systemically infected wheat plants inoculated with in vitro transcripts was confirmed by reverse-transcription polymerase chain reaction of the WSMV P3 gene and by accumulation of WSMV coat protein as detected by immunoblotting. Maintenance of the full-length WSMV cDNA in the high copy number plasmid pUC18 was problematic because of spontaneous rearrangement of WSMV sequences during growth in liquid media for more than 8 h or if the clone was subcultured. Stability of the WSMV cDNA clone was improved by the use of the low copy number plasmid pACYC177, and it could be grown in large scale volumes (up to 1 liter) of liquid culture for 14 h without noticeable rearrangements. Both the original WSMV culture and the progeny virus derived from infectious in vitro transcripts were efficiently transmitted by the natural eriophyid mite vector Aceria tosichella. This is the first report of infectious in vitro transcripts for any eriophyid mite-transmitted plant virus and represents the only monopartite member of the family Potyviridae infecting monocotyledonous hosts for which infectious in vitro transcripts are available.
摘要 使用全长 cDNA 克隆作为模板，用 SP6 RNA 聚合酶进行体外转录，产生了小麦线条花叶病毒（WSMV）的传染性 RNA。感染性取决于模板质粒 DNA 在大肠杆菌中扩增过程中是否发生自发重排。用体外转录物接种系统感染小麦植物时，WSMV 的存在通过 WSMV P3 基因的逆转录聚合酶链反应和免疫印迹检测到 WSMV 外壳蛋白的积累得到证实。由于在液体培养基中生长超过 8 小时或克隆被传代时，WSMV 序列会自发重排，因此将全长 WSMV cDNA 维持在高拷贝数质粒 pUC18 中存在问题。通过使用低拷贝数质粒 pACYC177，可以提高 WSMV cDNA 克隆的稳定性，并且可以在 14 小时内无明显重排地在 1 升大容量的液体培养基中生长。原始的 WSMV 培养物和源自传染性体外转录物的后代病毒都可以由天然的粉虱螨载体 Aceria tosichella 有效地传播。这是第一个关于任何粉虱螨传播植物病毒的体外传染性转录物的报告，也是唯一一种单分体家族 Potyviridae 感染单子叶宿主的成员，其具有可获得的体外传染性转录物。
