H. Suh et al., STRUCTURE OF THE AMPLIFIED 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE GENE IN GLYPHOSATE-RESISTANT CARROT CELLS, Plant molecular biology, 22(2), 1993, pp. 195-205
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.