Chloroplast targeting signal of a rice rbcS gene enhances transgene expression

Previously, we reported that the presence of the rbcS transit peptide (TP) sequence as a plastid-targeting signal led to greatly enhanced accumulation of transgene products in transgenic rice plants. To study the mechanisms underlying the regulation of transgene expression by the TP sequence, we mutated the sequence (mTP) so that it did not localize fused heterologous proteins into plastids, while it retained a nearly identical nucleotide sequence to its wild type. mTP was fused to a synthetic gene (sgfp) encoding a modified form of the green fluorescent protein (sGFP) and linked to the rice rbcS promoter, generating an expression vector, rbcS-mTP-sgfp. To compare expression levels in transgenic plants, two expression vectors were constructed: rbcS-sgfp for untargeted expression and rbcS-TP-sgfp for chloroplast-targeted expression. Several transgenic plants were produced for each construct by the Agrobacterium-mediated method. Confocal microscopic analyses demonstrated that GFP fluorescence in the rbcS-TP-sgfp-transformed lines was specifically localized within chloroplasts, whereas fluorescence was detected within cytoplasm and nucleoplasm in the rbcS-mTP-sgfp- and rbcS-sgfp-transformed lines. Northern blot analysis indicated that mRNA levels were very different, depending on the type of TP sequence employed. Transcript levels of rbcS-TP-sgfp-transformed lines were 10 fold higher than those of either rbcS-mTP-sgfp- or rbcS-sgfp-transformed lines, whereas the levels of the last two were comparable. Levels of sGFP accumulation were also correspondingly higher in rbcS-TP-sgfp-transformed lines than in the other two lines, as examined by immunoblot analyses. Thus, increased levels of sGFP accumulation appeared to reflect increased mRNA levels; this was due to the presence of the functional rbcS transit peptide. These results suggest that a functional rbcS transit peptide enhances transgene expression in addition to targeting the gene products into chloroplasts.