Figure 6.

Transgenic rice expressing GFP-labeled Rubisco. A, Schematic representation illustrating the OsRBCS2-sGFP fusion construct. Fusion proteins consisting of OsRBCS2 and sGFP were expressed under the control of the OsRBCS2 promoter. B to D, Protein analysis in transgenic rice expressing OsRBCS2-sGFP fusion. Total soluble proteins (10 μg for Coomassie Brilliant Blue R250 stain, 1 or 2 μg for immunoblotting, and 10 μg for detection of GFP fluorescence) extracted from shoots of plants expressing the OsRBCS2-sGFP fusion (RBCS2-sGFP), shoots of wild-type rice ‘Nipponbare’ plants as a control, and rGFP were separated by SDS-PAGE (B), nondenaturing PAGE using 4% to 12% gradient gels (C), or nondenaturing PAGE using 5% gels (D) and either stained with Coomassie Brilliant Blue R250 and subjected to immunoblotting with anti-GFP, anti-RBCS, or anti-RbcL antibodies or observed with a fluorescent image analyzer to detect GFP fluorescence. The white arrow indicates the mature form of RBCS2-sGFP after cleavage of the OsRBCS2 transit peptide, and the black arrow indicates the mature form of native OsRBCS after cleavage of its transit peptide. White arrowheads indicate GFP-labeled Rubisco holoenzyme (Rubisco-sGFP), and the black arrowhead indicates native Rubisco holoenzyme. The sizes of molecular mass markers (in kilodaltons) are indicated on the left of the stained gels. CBB, Coomassie Brilliant Blue R250; M. M., mass marker.