Immunological analysis of arabidopsis iandole-3-glycerol phosphate synthase

Indole-3-glycerol phosphate synthase (IGS, EC 4. 1. 1.48), one of the tryptophan biosynthetic pathway enzymes, catalyzes the conversion of 1-(O-carboxyphenoylamino)-1-deoxyribulose-5-phosphate (CDRP) into indole-3-glycerol phosphate, a precursor of indole ring-containing compounds including tryptophan, indole-3-acetic acid, phytoalexins and alkaloids. In order to study its gene expression, regulation and localization, an IGS cDNA isolated from Arabidopsis thaliana (L.) Heynh. cDNA library was constructed into an expression vector fused with glutathione S-transferase (GST) . After transforming into E. coli strain trpC9800λKC, the GST-IGS fusion protein was induced to overexpress by IPTG and purified with glutathione-agarose affinity chromatography and preparative SDS-PAGE. The purified GST-IGS fusion protein was used to immunize rabbit to raise polyclonal antisera. Western analysis of four Arabidopsis ecotypes, Columbia (Col-0), Landsberg erecta (Ler), Wassilewskija (Ws) and C24, identified a single polypeptide of approximate 40 kD. Accumulation of IGS was induced by stress treatments, indicating that IGS might play a role in plant defense system.