STRUCTURE OF THE AMPLIFIED 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE GENE IN GLYPHOSATE-RESISTANT CARROT CELLS

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for gly phosate resistance was analyzed to determine the mechanism of gene amp lification in this plant system. Southern hybridization of the amplifi ed DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in compariso n with the parental line. Cloning of the EPSPS gene and 5' flanking se quences was carried out and two different DNA structures were revealed . A 13 kb clone contained only one copy of the EPSPS gene while a 16 k b clone contained an inverted duplication of the gene. Southern blot a nalysis with a carrot DNA probe showed that only the uninverted repeat ed DNA structure was present in all of the cell lines during the selec tion process and the inverted repeat (IR) was present only in highly a mplified DNA. The two structures were present in about equal amounts i n the highly amplified line, TC 35G, where the EPSPS gene was amplifie d about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation an d rapid renaturation, showing foldback DNA with the IR length being 9. 5 kb. The junction was also sequenced. Mapping of the clones showed th at the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.