Fully biologically active in vitro transcripts of the eriophyid mite-transmitted wheat streak mosaic tritimovirus
Fully biologically active in vitro transcripts of the eriophyid mite-transmitted wheat streak mosaic tritimovirus
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DOI：
10.1094/phyto.1999.89.12.1182
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发表时间：
1999-12-01
期刊：
影响因子：
3.2
通讯作者：
Stenger, DC
中科院分区：
文献类型：
--
作者：
Choi, IR;French, R;Stenger, DC
以小麦条斑花叶病毒（WSMV）的全长cDNA克隆为模板，用SP 6 RNA聚合酶进行体外转录，获得了WSMV的侵染性RNA。亲和性依赖于在大肠杆菌中扩增过程中未发生自发重排的模板质粒DNA的使用。WSMV P3基因的逆转录-聚合酶链反应和WSMV外壳蛋白的积累，通过免疫印迹检测，证实了WSMV在系统感染的小麦植株接种在体外成绩单的存在。在高拷贝数质粒pUC 18中的全长WSMV cDNA的维持是有问题的，因为在液体培养基中生长超过类似于8小时或如果克隆被继代培养期间WSMV序列的自发重排。通过使用低拷贝数质粒pACYC 177，WSMV cDNA克隆的稳定性得到改善，并且它可以在大规模体积（高达1升）的液体培养物中生长类似于14小时而没有明显的重排。原始WSMV培养物和感染性体外转录产物衍生的子代病毒均能通过天然瘿螨载体Aceria tosichella有效传播。这是第一次报告的感染性体外转录的任何eriophyid螨传播的植物病毒，并代表唯一的monopartite成员的家庭Potyviridae感染单子叶植物的主机，其中感染性的体外转录可用。
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 similar to 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 I liter) of liquid culture for similar to 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.