Subcellular targeting of green fluorescent protein to plastids in transgenic rice plants provides a high-level expression system

• Published in: Molecular breeding Vol. 5; no. 5; pp. 453 - 461
• Main Authors: Jang, I.C, Baek Hie Nahm, Kim, J.K
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Nature B.V 1999
• Subjects: Agrobacterium; Agronomy; animal proteins; Cell fusion; Chloroplasts; cytochemistry; Cytoplasm; Expression vectors; Fluorescence; Fusion protein; Gene expression; gene transfer; genetic transformation; Green fluorescent protein; Leaves; Levels; Molecular biology; Oryza; Oryza sativa; Plant biology; Plant tissues; Plastids; protein transport; Proteins; recombinant proteins; ribulose-bisphosphate carboxylase; Rice; Scyphozoa; sgfp gene; signal peptide; synthetic genes; Transgenic plants
• ISSN: 1380-3743; 1572-9788
• DOI: 10.1023/A:1009665314850

In order to develop a high-level expression system in transgenic rice, we inserted a synthetic gene (sgfp) encoding a modified form of the green fluorescent protein (GFP) into two expression vectors, Act1-sgfp for an untargeted and rbcS-Tp-sgfp for a chloroplast targeted expression. Several fertile transgenic rice plants were produced by the Agrobacterium-mediated method. Confocal microscopic analyses demonstrated that, in cells expressing the Act1-sgfp, GFP fluorescence was localized within the cytoplasm and nucleoplasm whereas, in cells expressing the rbcS-Tp-sgfp fusion gene, the fluorescence was specifically targeted to chloroplasts and non-green plastids. The levels of sgfp expression were about 0.5% of the total soluble protein in mature leaf tissues of the Act1-sgfp transformed lines. In contrast, expression levels were markedly increased in mature leaf tissues of the rbcS-Tp-sgfp transformed lines, yielding about 10% of the total soluble protein. N-terminal sequencing of the localized GFPs revealed that the Tp-GFP fusion protein was correctly processed during import to non-green plastids, as well as to chloroplasts. Thus, our results demonstrate that GFP can be produced at high levels and localized in specific subcellular spaces of transgenic plants, providing a high-level expression system for general use in rice, an agronomically important cereal.