Identification of a new gene encoding 5-enolpyruvylshikimate-3-phosphate synthase using genomic library construction strategy

摘要

Applying the genomic library construction strategy and colony screening, a new aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase has been identified, cloned and overexpressed in Escherichia coli, and the enzyme was purified to homogeneity. Kinetic analysis of the AroA P.fluorescens indicated that the full-length enzyme exhibits 10-fold increased IC50 and an approximately 38-fold increased K i for glyphosate compared to those of the AroA E.coli , while retaining high affinity for the substrate phosphoenolpyruvate. Furthermore, we have transformed the new aroA P.fluorescens gene into Arabidopsis thaliana via a floral dip method, and demonstrated that transgenic A. thaliana plants exhibit significant glyphosate resistance when compared with the wild type.