Dicistronic expression of the green fluorescent protein and antibiotic resistance genes in the plastid for selection and tracking of plastid-transformed cells in tobacco

Abstract

A plastid transformation vector was constructed for dicistronic expression of the aminoglycoside 3′-adenyltransferase (aadA) and green fluorescent protein (gfp) genes under the control of the plastid rrn promoter. Gold particles coated with the vector DNA were bombarded onto tobacco leaf explants using a particle delivery system. Leaf explants produced adventitious shoots when cultured on shoot-inducing medium containing 500 mg 1-1 spectinomycin. Shoots that exhibited green fluorescence under UV light were selected. Southern blot analysis detected the presence of the aadA and gfp genes between trnA and trnI in the plastid genome. Northern blot analysis revealed that the aadA and gfp genes were both properly transcribed into a dicistronic transcriptional unit. The expression of the gfp gene in the plastid enabled separation of transformed chloroplasts from wild-type chloroplasts in the protoplast under a fluorescent microscope. The overall results indicate that dicistronic expression of the aadA and gfp genes in the plastid simplifies gene manipulation, facilitating selection and tracking of plastid-transformed cells.