Study On The Effects Of Ectopic Expression Of A Glutamate Dehydrogenase Gene From Eurotium Cheralieri On The Nitrogen Metabolism In Rice(Oryza Sativa L.)

Nitrogen is one of the necessary mineral element of plant for growth and development. Most plants can take up the inorganic nitrogen as the form of NH4+and NO3-by root or produce by metabolize in vivo. All of those inorganic nitrogen must be converted into organic nitrogen in order to use for plants. There are two pathways of ammonium assimilation in plants:GS/GOGAT and the GDH pathway. GDH enzymes in plants are known to exhibit higher Km values for NH4+, that is to say, GDH has a lower affinity with NH4+. As a result, the NH4+can’t be effective utilized. However, assimilation of NH4+via GDH consumes less energy compared with the GS/GOGAT. Therefore, increasing the efficiency of GDH may be one of the better ways to improve the nitrogen use efficiency of plant. Rice (Oryza sativa L.) is an important food crop in our country. Nitrogen constitutes the large part of rice production cost, while the average utilization rate of nitrogen is about only28%-37%. In this study, the GDH gene(EcGDH) was cloned from Eurotium cheralier in order to explore a possible way to improve nitrogen utilization rate of rice. Some related researches were conducted on EcGDH, and the main results of this study are as follows:1. Homonlogy and phylogenetic analysis showed that GDH of Eurotium cheralier has a higher homology with the GDH of Nigrospora, but with a lower homology with the OsGDH. What’s more, amino acid sequence aligement indicated that EcGDH belonged to NADPH-GDH, and the protein encoded by EcGDH was a stable hydrophobic protein.2. The EcGDH gene cloned from Eurotium cheralier was about1380bp, the sequencing result was as the expected.3. The prokaryotic expression vector pCold-EcGDH had been constructed, and the expression conditions were optimized. The optimal condition for prokaryotic expression was performed at16℃for12h with0.2mmol/L IPTG. The specificity of purified recombinant protein EcGDH was confirmed by Western blotting. Further more, the enzymatic activity of recombinant protein EcGDH had been detected by spectrophotometry in vitro. The Km of EcGDH with NH4+and a-oxoglutarate(a-OG) were6.06±0.33mmol/L,8.13±0.45mmol/L respectively, which meant EcGDH had the enzymatic activity of glutamate dehydrogenase.4. By constructing over-expression vector pCAMBIA1301-EcGDH to transfo- rm rice, stable transgenic lines were generated through agrobacterium-mediated t-ransformation. Moreover, Over-expression of Ubi::EcGDH transgenic lines Ubi::EcGDH-12and Ubi::EcGDH-13were obtained through the resistance selecti-on of hygromycin and molecular identification.5. The hydroponic experiment of positive transgenic lines was carried out under two different NH4NO3concentration(200μmol,2000μmol) conditions. The results indicated that the total nitrogen content was increased12.7%in Ubi::EcGDH transgenic lines compared with non-transgenic in lower nitrogen concentration. Meanwhile, the enzymatic activity of NADP (H)-GDH in transgenic plants had been detected and the results showed that the NH4+assimilation in rice had been promoted by introduce an exogenous gene EcGDH. The field trials indicated that the plant height, panicle number and grain yield of Ubi::EcGDHtransgenic plants increased in varying degrees, eapecially the plant height increased significantly. Whereas, there were no obvious differences in1000-grain weight and seed-setting rate between the transgenic lines and the control plants. The nitrogen utilization rate of rice had been influenced in some ways although the transgenic lines with distinct advantages in growth and yeild had not been obtained.