Setor de Fitossanidade/Centro de Ciências Agrárias, Universidade Federal de Alagoas, Rio Largo, AL, 57100-000, Brazil.
Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
J Virol Methods. 2019 Jan;263:20-23. doi: 10.1016/j.jviromet.2018.10.017. Epub 2018 Oct 23.
Viruses belonging to the genus Begomovirus (family Geminiviridae) have circular single-strand DNA genomes encapsidated into quasi-icosahedral particles, and are transmitted by whiteflies of the Bemisia tabaci complex. Biological and molecular properties of begomoviruses have been studied efficiently with infectious clones containing dimeric genomic components. However, current approaches employing enzymatic digestion and ligation to binary vectors are laborious, mostly due to many cloning steps or partial digestion by restriction enzyme. Here, an infectious clone of the bipartite begomovirus Bean golden mosaic virus (BGMV) was obtained using PCR and Gibson Assembly (GA). Common bean (Phaseolus vulgaris) seedlings displayed severe yellow mosaic and stunt symptoms 15 days after agroinoculation with DNA-A and DNA-B of BGMV. The approach based on PCR-GA protocol is a fast and useful tool to obtain infectious clones of a circular DNA plant virus.
属于 BGMV 属的病毒（家族 Geminiviridae）具有环形单链 DNA 基因组，被包裹在准二十面体颗粒中，并通过烟粉虱复合种传播。使用包含二聚体基因组成分的传染性克隆，已经有效地研究了 BGMV 的生物学和分子特性。然而，目前采用酶切和连接到二元载体的方法非常繁琐，主要是由于克隆步骤多或部分受限制酶消化。在这里，使用 PCR 和 Gibson 组装（GA）获得了二分体 BGMV（Bean golden mosaic virus）的传染性克隆。在农杆菌接种 BGMV 的 DNA-A 和 DNA-B 后 15 天，普通菜豆（Phaseolus vulgaris）幼苗表现出严重的黄色花叶和矮化症状。基于 PCR-GA 方案的方法是获得环状 DNA 植物病毒传染性克隆的快速有用工具。
