Figure 1.

Construction of a yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector: (a) Schematic diagram illustrating construction of the shuttle plasmid pCB301-2μ-HDV. A fragment containing the yeast 2μ replication origin and TRP1 selection marker gene was amplified from the yeast plasmid pGBK-T7 with primers TRP/R and 2μ-ori/F and then inserted into the pCB301-HDV Agrobacterium binary vector through the AfeI restriction site. p35S², doubled CaMV 35S promoter; (b) Transient expression of eGFP in agroinfiltrated leaf patches. Nicotiana benthamiana leaves were infiltrated with Agrobacterium mixtures carrying the pCB301-2μ-eGFP or pCB301-eGFP plasmid and the TBSV p19 suppressor plasmid. The infiltrated leaves were visualized under a fluorescence microscope at 36 h post infiltration; (c) Total proteins extracted from infiltrated leaf patches were analyzed in a Western blot with an eGFP-specific antibody. TRP1: tryptophan selection maker gene; OriV: RK2 replication origin; TrfA: replication initiator protein; HDRz: HDV ribozyme; tNos: Nos terminator; 2μ ori: yeast 2μ replication origin; KanR: kanamycin resistance gene; LB: left border sequence; RB: right border sequence; eGFP: enhanced green fluorescence protein.