Junqueira Bruna Rayane Teodoro, Nicolini Cícero, Lucinda Natalia, Orílio Anelise Franco, Nagata Tatsuya
Pós-graduação em Biologia Molecular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil.
Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil.
J Virol Methods. 2014 Mar;198:32-6. doi: 10.1016/j.jviromet.2013.12.024. Epub 2013 Dec 31.
Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants.
RNA病毒的感染性cDNA克隆是研究复制和宿主-病毒相互作用等分子过程的重要工具。然而，在二元载体中构建病毒RNA基因组cDNA所需的克隆步骤通常很繁琐。在本研究中，详细描述了一种生产农杆菌感染性辣椒轻斑驳病毒（PMMoV）克隆的简化方法。最初，通过一步法RT-PCR扩增PMMoV的完整基因组，克隆并亚克隆到T7 RNA聚合酶启动子下的小质粒载体中，通过转录接种确认cDNA克隆的感染性。然后使用一步重叠延伸PCR将完整基因组转移到二元载体中。将筛选出的克隆农杆菌浸润到本氏烟草植株中，结果显示具有感染性，可引起典型的PMMoV症状。当将野生型PMMoV分离株、T7 RNA聚合酶衍生的转录本和农杆菌浸润衍生的病毒接种到本氏烟草、中国辣椒PI 159236和辣椒植株上时，未观察到宿主反应的差异。
