Construction of a plant expression vector for the coat protein gene of cucumber mosaic virus-As strain for plant transformation

Ryu KiHyun; Park WonMok

Korean Journal of Plant Pathology 11(1): 66-72


Accession: 002787386

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The coat protein (CP) gene of cucumber mosaic cucumovirus As-strain (CMV-As) was engineered for expression using cauliflower mosaic caulimovirus 35S transcript regulatory sequences. The CP gene was cloned into an Agrobacterium-derived binary vector. A chimeric gene was constructed by the cDNA of CMV-As CP and plant expression vector pBI121. The clone pCMAS66 was first introduced into the phagemid vector pSPORT1 for situating sense orientation for translation and making restriction sites to re-introduce plant expression vector, pBI121. The resulting subclone pCASCP02 and plant expression vector pBI121, were treated with BamHI-SacI for excising the target gene and removing the GUS gene, respectively. After Agrobacterium transformation by freeze-thawing, the clone pCMASCP121-123 which contains sense orientation of the target gene, was selected and confirmed by restriction endonuclease analysis. The CMV-As CP gene was introduced into A. tumefaciens. Transformation of tobacco with this vector system was successful and showed high frequency of selection of putative transformants.