Baculovirus–mediated gene delivery into plant cells

Baculovirus has been widely used for production of numerous recombinant proteins in insect cells. Baculovirus expression has the following advantages: proper post-translational modification, a high capacity for expression of multiple genes or a large insert, biosafety, and high yield driven by the strong promoters polyhedrin or p10. Recently, recombinant proteins have been expressed in both insect cell and mammalian cells by baculovirus system. To date, however, there has been no report of recombinant protein expressed by baculovirus infection in plant. In this study, this expression system was exploited to express GFP (green fluorescence protein). To investigate the baculovirus capability to express the recombinant protein in plant cell, the GFP was cloned under the control of polyhedrin (PPH) promoter and 35S (Cauliflower Mosaic Virus) promoter into the pFastBacTM dual vector, respectively. Gene expression cassetees carrying the GFP genes were transposed into a bacmid in Escherichiacoli (DH10Bac). Bacmid DNA containing GFP gene expression cassette was isolated from transformed DH10Bac using Bac-to-Bac system. The existence of GFP gene in bacmid was confirmed by PCR with GFP specific primers. Both bacmid and baculovirus generated from bacmid-infected Sf9 cells was transfected NicotianaBenthamiana plant leaves, respectively. Confocal immunoflouorescence and fluorescence microscopic analyses confirmed expression of GFP in baculovirus-infected plant leaves and protoplasts isolated from baculovirus–infected plant leaves. In additon, reverse transcription-polymerase chain reaction (RT-PCR) confirmed transcription of GFP gene. In this study, taken together, these results suggest that the baculovirus is able to express recombinant protein gene in plant cells.