The Effects Of Fungal GDH Gene (CeGDH) Expression In Rice (Oryza Sativa L.) On Nitrogen Utilization

Glutamic acid deaminase (GDH) is widespread in the organism. GDHs in higherplants have lower affinities for ammonium, so their nitrogen is mainly assimilated asammonium by the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway.In lower organisms, NADP(H)-dependent glutamate dehydrogenases (GDHs) havehigh affinities for ammonium and play important roles in nitrogen assimilation. GDHsfrom lower organisms were introduced into plants so as to modify the ammoniumassimilation. Rice is one of the most important crops in the world, as it is the primaryfood source for more than half of the world’s population. Some related researcheswere conducted on CeGDH, and the main results of this study are as follows:1. Those phylogenetic analysis results revealed that CeGDH aNADP(H)-dependent glutamate dehydrogenase gene from fungus Cylindrocarponehrenbergii has a low homology with the GDH amino acid sequences which fromOryza sativa and Arabidopsis thaliana, and has a high homology with gdhA the aminoacid sequence of Aspergillus niger GDH and NiGDH the amino acid sequence ofNeurospora intermedia GDH.2. In present study, CeGDH was cloned and ectopic expressed in rice (Oryzasativa cv. Kitaake). Construction of recombinant prokaryotic expression vectorpCold-TF-CeGDH，then the CeGDH was expressed on the induction of IPTG，a100kDa band was abserved. The NADP(H)-GDH activity in vitro was determined byusing the purified CeGDH. The determination of enzymatic activity in vitro showedthat CeGDH aminating activity (3.24μmol min-1mg-1) is higher than deaminatingactivity (0.28μmol min-1mg-1).3. A CeGDH-YFP fusion protein was expressed transiently in Arabidopsisprotoplasts (35S::CeGDH-YFP). The results illustrated that CeGDH is mainlylocalized in the cytoplasm where ammonium assimilation takes place.4. The over-expression vector pCAMBIA1301GW-CeGDH was introduced intorice by Agrobacterium (EHA105)-mediated transformation method. Transgenic plantswere determined by RT-PCR and Western Blotting.5. The total proteins of transgenic lines (Ubi::CeGDH-7and Ubi::CeGDH-33)and WT were extracted used for activities assay. The result of enzymatic activity invivo in CeGDH transgenic rice was also confirmed by the results of enzymatic activityin vitro. The activities assay of NADPH-dependent enzyme activities in plants show that aminating activities of WT，Ubi::CeGDH-7，Ubi::CeGDH-33were0.277，0.310，0.337μmol min-1mg-1respectively, while deaminating activities were0.0452，0.0217，0.0250μmol min-1mg-1respectively. These results suggested that the CeGDHis inclined to interconvert2-Oxoglutarate to glutamate.6. The experiment of hydroponics revealed that the shoot and root length andnitrogen content were improved obviously in transgenic plants at seedling stage,compared with the wild type under low-nitrogen conditions. In field condition, theagronomic traits analysis showed that, in comparison with the wild type, the paniclenumbers and grain yields of transgenic plants also increased significantly inlow-nitrogen conditions.These results demonstrated that the introduction of CeGDH in rice can enhancerice nitrogen utilization，improve its growth at seedling stage and grain yields.