National Institute of Genetic Engineering and Biotechnology (NIGEB
The engineering of transgenic canola (Brassica napus L. ) to make tolerance to the broad-spectrum herbicide, glyphosate, is one of the most effective approaches for weed management. Glyphosate inhibits the enzyme EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) enzyme which functions in the shikimate pathway and has a key role in biosynthesis of aromatic amino acids required for survival of the plant. Induction of glyphosate tolerance in transgenic canola via introducing mutated epsps to the plant genome has been previously reported. By this strategy, enzyme’s affinity for glyphosate is reduced. Applying glyphosate degrading enzyme of bacterial origin such as glyphosate oxidoreductase (GOX) in combination with a glyphosate-tolerant epsps is the ultimate approach to provide commercial rates of glyphosate tolerance. In this project, a synthetic geneencoding GOX enzyme with plant codon preferences was designed. The structure of the synthetic construct and its mRNA were analyzed by bioinformatic tools. This synthetic gene was subcloned and transformed into canola plant via Agrobacterium mediated transformation in order to investigate the potential roles in increasing glyphosate tolerance. The presence, copy numbers and expression of the transgene were confirmed by PCR, Southern blotting and RT-PCR analyses, respectively. The bioassay with glyphosate challenging showed that the transgenic plant tolerated glyphosate at a concentration of 1.5 mM whereas the non-transformed canola was unable to survive in the presence 0.5 mM glyphosate.