Figure 2. Expression of PAL in transgenic rice seeds.

(A) Analysis of total protein extracted from transgenic rice seeds by SDS-PAGE and by immunoblot using anti-PAL antibody, anti-PAL. WT, non-trangenic rice; and M, Precision plus protein Standards (Bio-Rad). Empty trangle denoted the band of synthesized EiP in SA seeds and black triangle denoted the immunoactive band of N-glycosylated PAL. Arrows denoted PAL and ELP-intein-PAL fusion protein (EiP). (B) N-Glycosylation analysis of PAL in SP (top panel) and EiP in SA seeds (bottom panel) by Endo H digestion. gPAL, N-glycosylated PAL and gEiP, N-glycosylated EiP. (C) Detection of Ei tag in total protein extracted from SA seeds by immunoblot using anti-ELP antibody. Only EiP fusion protein was detected while free Ei tag was not observed. Arrows denoted N-glycosylated PAL (gPAL), PAL and ELP-intein-PAL fusion protein (EiP). (D) Immunoblot analysis on total protein extracted from SA seeds of different trnagenic lines (1–6) using anti-PAL antibody. (E) Immunoblot analysis of recombinant proteins extraction from transgenic rice seeds by four different extraction buffers, B1, B2, B3 and To. Total protein samples extracted from equal amount of rice seed powder by different buffers (see Materials and Methods) were used for analysis. (F) Relative accumulation levels of PAL in SP and SA rice seeds. gPAL, N-glycosylated PAL; and ePAL, relative amount of PAL derived from ELP-intein-PAL fusion protein. (G) Accumulation levels of uncleaved EiP in T1 and T2 seeds of SA transformed rice. Error bars indicate standard deviation among different transgenic lines.