Chimeric genes as dominant selectable markers in plant cells

Abstract

Opine synthases are enzymes produced in dicotyledonous plants as the result of a natural gene transfer phenomenon. Agrobacteria contain Ti plasmids that direct the transfer, stable integration and expression of a number of genes in plants, including the genes coding for octopine or nopaline synthase. This fact was used as the basis for the construction of a number of chimeric genes combining the 5' upstream promoter sequences and most of the untranslated leader sequence of the nopaline synthase (nos) gene with the coding sequence of two bacterial genes: the aminoglycoside phosphotransferase (APH(3')II) gene of Tn5 and the methotrexate-insensitive dihydrofolate reductase (DHFR Mtx) of the R67 plasmid. The APH(3')II enzyme inactivates a number of aminoglycoside antibiotics such as kanamycin, neomycin and G418. Kanamycin, G418 and methotrexate are very toxic to plants. The chimeric NOS-APH(3')II gene, when transferred to tobacco cells using the Ti plasmid as a gene vector, was expressed and conferred resistance to kanamycin to the plant cells. Kanamycin-resistant tobacco cells were shown to contain a typical APH(3')II phosphorylase activity. This chimeric gene can be used as a potent dominant selectable marker in plants. Similar results were also obtained with a NOS-DHFR Mtx gene. Our results demonstrate that foreign genes are not only transferred but are also functionally expressed when the appropriate constructions are made using promoters known to be active in plant cells.