Fig 1.
Maps of vectors and sequences of linker peptides.
(A) Maps of the vectors used to analyze cleavage efficiency. (B) Maps of the vectors used to analyze subcellular location. (C) DNA and corresponding amino acid sequences of various linker peptides. The underlined sequences encode amino acids APVK and SGSG, which were added to improve cleavage efficiency. 35S: Cauliflower mosaic virus 35S promoter; UBI: Ubiquitin promoter; GFP: green fluorescent protein; RFP: red fluorescent protein; GUS: β-Glucuronidase; NOS: Nopaline synthase gene terminator; ER: endoplasmic reticulum signal peptides; Chl: chloroplast signal peptides; 2A, LP4, and LP4/2A: linker peptides.
Fig 2.
GUS analyses of different vector transformed protoplasts.
(A) GUS histochemical assay of different linker peptides in transformed protoplasts. (B) GUS enzyme activity of different linker peptides in transformed protoplasts. The transformed protoplasts were processed for the GUS fluorometric assay 24-h post-transformation. The mean and standard deviation of GUS enzyme activity in each transformed protoplast are presented.
Fig 3.
GFP analyses of different vector transformed protoplasts.
(A) Confocal microscopy of the transformed protoplasts. Green signals indicate cleaved GFP. The scale bar represents 100 μm. (B) Quantification of GFP expression. The amount GFP expression in each form of vector was estimated from its fluorescence intensity measured by Image J software.
Fig 4.
Cleavage efficiency analyses of different vector transformed protoplasts.
(A) Western blot analysis of cleavage efficiency of the linker peptide in protoplasts. The transformed protoplasts were processed for Western blot 24-h post-transformation. The cleavage efficiency was assessed using GFP, GUS and 2A antibodies. Anti-β-actin antibody was used as a loading control. Asterisks represent uncleaved proteins. (B) Quantification of the cleavage efficiency of different linker peptides. Cleavage efficiency = cleaved form/(cleaved form + uncleaved form). The amount of each form was estimated from its band intensity on the western blot measured by Image J software.
Fig 5.
Protoplasts transformed by subcellular locational vectors.
(A) Protoplasts transformed by pUg and pUr. A: GFP excitation at 488 nm and RFP excitation at 543 nm; B: light field; and C: AB merge; scale bar = 10 μm. (B) Protoplasts transformed by pUgLr. A: GFP excitation at 488 nm; B: RFP excitation at 543 nm; C: light field; and D: ABC merge; scale bar = 10 μm. (C) Protoplasts transformed by pUCgLr and pUrLCg. A: GFP excitation at 488 nm; B: RFP excitation at 543 nm and emission at 550–710 nm; C: RFP excitation at 543 nm and emission at 550–610 nm (no chlorophyll auto-fluorescence); D: light field; and E: ABD merge; scale bar = 10 μm. (D) Protoplasts transformed by pSEb and pUEg. A: BFP excitation at 405 nm and GFP excitation at 488 nm; B: light field; and C: AB merge; scale bar = 10 μm. (E) Protoplasts transformed by pUEgLr, pUgLEr, pUCgLEr and pUErLCg (co-transformed with pSEb). A: excitation at 488 nm; B: excitation at 543 nm; C: excitation at 543 nm; D: light field; and E: ABCD merge; scale bar = 10 μm.
Table 1.
The report proteins’ sublocalization after protoplast transformations with different vectors.
Table 2.
The summary of the prospective proteins and their molecular weights.
Fig 6.
Western blot analysis of report genes expression in subcellular locational vectors.
Transformed protoplast was analyzed by western blot to check proteins and their molecular weight, and the antibody is anti-GFP. GFP CK+: GFP positive control; pUg: protoplast transformed by pUg; pUCgLr: protoplast transformed by pUCgLr; pUrLCg: protoplast transformed by pUrLCg; pUCgLEr: protoplast transformed by pUCgLEr; pUErLCg: protoplast transformed by pUErLCg; pUEgLr: protoplast transformed by pUEgLr; pUEg: protoplast transformed by pUEg; pUEgLr: protoplast transformed by pUEgLr; pUgLEr: protoplast transformed by pUgLEr; pUgLr:protoplast transformed by pUgLr.
Fig 7.
Western blot analysis of RFP expression in transformation protoplast (anti-RFP).
Transformed protoplast was analyzed by western blot to check proteins and their molecular weight and the antibody is anti-RFP. RFP CK+: RFP positive control; pUgLr: protoplast transformed by pUgLr; pUCgLr: protoplast transformed by pUCgLr; pUrLCg: protoplast transformed by pUrLCg; pUEgLr: protoplast transformed by pUEgLr; pUgLEr: protoplast transformed by pUgLEr; pUCgLEr: protoplast transformed by pUCgLEr; pUErLCg: protoplast transformed by pUErLCg.