Molecular cloning, characterization and function analysis of a GDH gene from Sclerotinia sclerotiorum in rice

The full-length cDNA encoding a glutamate dehydrogenase (GDH) which catalyzes the reaction of reductive amination of α-oxoglutarate (α-OG) to glutamate (the anabolic activity) and the reverse reaction of oxidative deamination of glutamate (the catabolic activity) was isolated from Sclerotinia sclerotiorum, we designated it as SsGDH. Bioinformatics analysis revealed that SsGDH had a typical GDH spatial structure and extensive homology with other fungal or bacteria GDHs. To evaluate its function in rice, rice (Oryza sativa L. cv. 'kitaake') was transformed with SsGDH in a binary vector construct by Agrobacterium-mediated transformation. Transgenic rice plants showed that transcripts and proteins of SsGDH accumulated at higher levels and GDH enzymatic activity was obviously higher in transgenic rice plants compared with the non-transformant rice plants (CK), though phenotype including plant height, fresh weight and dry weight became slightly weaker compared with CK under 50, 500 and 5,000 μM nitrogen gradient nutrient solution treatment (NH4NO3 as a nitrogen source) after introducing SsGDH into rice. For enzymatic activity assay in vitro, recombinant His6-SsGDH protein was expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA agarose. Results suggested that recombinant His6-SsGDH protein had GDH activity using ammonium, α-OG, and L-glutamate separately as a substrate at two different concentrations, especially the affinity for ammonium was very high, and its Km value was only 0.28 ± 0.03 mM, indicating that SsGDH can assimilate more ammonium into rice. According to previous reports, transgenic plants expressing fungal or bacteria GDHs might show improved herbicide resistance. Basta resistance test showed that SsGDH expression in rice can significantly enhanced their tolerance to Basta than CK. In conclusion, our results may provide some clues for further investigation on nitrogen utilization via introducing exogenous GDHs from lower organisms into rice.