XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta

Posters
13.
EXPRESSION, PURIFICATION AND FUNCTIONAL CHaraCTERIZATION
OF TWO FORMS OF agroBACTerIUM SP. STraIN CP4 EPSPS GENE IN
ESCherICHIA COLI FOR HORIZONTAL GENE TraNSFER STUDIES
Mahesh Madyagol, Stanislav Stuchlík and Jan Turňa
Comenius University, Department of Molecular Biology, Faculty of Natural Sciences,
Bratislava, SLOVAKIA
The Agrobacterium sp. strain CP4 aroA gene encodes an enzyme called 5-enol-pyruvylshikimate-3-phosphate
(EPSP) synthase. The enzyme is widely involved in glyphosate
tolerant transgenic plants because it is the primary target of the nonselective herbicide
glyphosate. We have cloned this gene and constructed a system for the high level expression
of a recombinant form of this enzyme by amplifying the aroA gene from the
genetically modified maize genomic DNA and the coding sequence of EPSPS gene was
successfully subcloned in a plasmid-Escherichia coli system. Furthermore, the truncated
form of CP4 EPSPS synthase has been created in order to compare in vitro glyphosate
sensitivity between the two forms of enzymes. The cells containing the plasmid carrying
both forms of EPSPS gene exhibited enhanced tolerance to herbicide glyphosate,
compared to the control. The resulting plasmids produced the EPSP synthase in large
quantities which has been purified to homogeneity and enzyme activity assay has been
carried out. The study of horizontal gene transfer (HGT) is planned in the future work.
128 XXII. Biochemistry Congress, Martin