Journal
JOURNAL OF VIROLOGICAL METHODS
Volume 222, Issue -, Pages 11-15Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jviromet.2015.05.003
Keywords
Gibson Assembly; Seamless cloning; Full-length cDNA clone; Tymovirus
Funding
- National Council of Technological and Scientific Development [CNPq 479161/2013-5]
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The construction of full-length infectious clones of RNA viruses is often laborious due to the many cloning steps required and the DNA exclusion within the plasmid during Escherichia coli transformation. We demonstrate single-step cloning procedure of an infectious cDNA of the tomato blistering mosaic virus (ToBMV) using Gibson Assembly (GA), which drastically reduces the number of cloning steps. By agro-inoculation with the construct obtained by this procedure, ToBMV was recovered six days post-inoculation in Nicotiana benthamiana plants. The symptoms induced by the recovered virus were indistinguishable from those caused by the wild-type virus. We conclude that the GA is very useful method particularly to construct a full-length cDNA clone of a plant RNA virus in a binary vector. (C) 2015 Elsevier B.V. All rights reserved.
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