Molecular Mechanism Of Glutamine Dehydrogenase Genes For Primary Study Of The Rice Nitrogen Metabolite

Nitrogen is a crucial plant macronutrient and is needed in the greatest amount of all mineral elements required by plants.It is a constituent of numerous important compounds, including amino acids,proteins,nucleic acids,chlorophyll and several plant hormones. Nitrogen cycle is a complex cycle in the plant system,involving many enzymes.And the ammonium taken up by plant root or produced by reduction of nitrate is first assimilated by glutamine synthetase(GS;EC 6.3.1.2) to yield the amino group of glutamine(Gln) in an ATP-dependent reaction.GS is coupled with glutamate synthase(GOGAT; Fd-GOGAT:EC 1.4.7.1 and NADH-GOGAT:EC 1.4.1.14) in a so-called GS/GOGAT cycle.GS produces Gln from ammonium and glutamate(Glu),and GOGAT transfers the amino group of Gln toα-oxoglutarate(α-OG) to generate two molecules of Glu in the cycle.Alternatively,ammonium is incorporated into Glu by the reversible reductive amination ofα-OG,which is catalyzed by glutamate dehydrogenase(GDH;1.4.1.2～4). The GS/GOGAT pathway is regarded as the primary pathway for ammonium assimilation, while the function of GDH pathway remains obscure.Assimilation of NH₄⁺ via GDH confers a saving in energy compared with the GS/GOGAT cycle.Thus,increasing the efficiency of GDH is one of the better ways to increase the nitrogen use efficiency of the plant system according to energy penalty.Rice(Oryza sativa L.) is an important and model cereal crop for functional genomics research.In this study,a Chlorella sorokiniana gdhANC gene was overexpressed by CaMV35S and Ubiquitin promoter in rice plants,the phenotypes were analysed in transgenic plants by molecular biology and physiology methods,in order to find the transgenic rice plant which exhibited high nitrogen use efficiency.At the same time,gdh genes in the rice,including OsGDH1 and OsGDH4, were studied for their function in the nitrogen by overexpressing,silencing or derivative of the gene promoter EGFP.In addition,a molecular approach is needed to characterize GDHs in the rice species by analyzing the structure and expression of this gene family.It is expected that such a comprehensive analysis may provide a framework for future functional dissection of the GDH gene family in plant growth and development.The main results are as follows:1.There are four putative GDH genes(OsGDH1～4) in the rice genome.The GDH sequences from rice and other species can be classified into two types(â… andâ…¡). OsGDH1～3 belonged to typeâ…¡genes whereas OsGDH4 belonged to typeâ… like gene. Our data implied that the expansion rate of typeâ… genes was much slower than that of typeâ…¡genes and species-specific expansion contributed to the evolution of typeâ…¡genes in plants.2.Gene expression patterns revealed that OsGDH1,OsGDH2 and OsGDH4 are expressed ubiquitously in various tissues/organs,whereas OsGDH3 expression is glumes and stamens specific.3.The expression of the OsGDH family members responded differentially to nitrogen and phosphorus deprivation,indicating their roles under such stress conditions.4.The results of hierarchical cluster analysis(HCA) indicate only two GDH genes (OsGDH2 and OsGDH4) are clustered with GS and GOGAT genes.Although we can yet not determine the in vivo direction of individual GDH genes based on the HCA,results from the HCA indicate non-redundant functions among OsGDH family members under the normal conditions.5.Based on the p1301S vector which contained CaMV35S promoter,three constructs overexpressed OsGDH1,OsGDH4 and gdhANC respectively,were transformed into rice plants.The copy numbers and expression level of transformed genes were also checked by Southern and Northern blot respectively.And the phenotype and biomass of these genes-overexpressed plants exhibited no significant changes when compared with wild type plants.However,the results of biochemical and physiological testing showed a higher metabolic level in the transgenic plants compared with wild type plants,including GDH activities in leaves and nitrogen content in seeds.Replacing CaMV35S promoter, we used Ubiquitin promoter to overexpress gdhANC in rice plants and similar results were obtained.6.The results of abiotic stress indicated gdh-overexpressed plants at heading stage showed a significantly high resistance to high concentration of NaCl(200 mmol/L) compared to the wild-type plants.