The Application Of Fungal Glutamate Dehydrogenase In Improving The Nitrogen Usage Efficiency Of Crops

Nitrogen is an essential macroelement for plant growth and development, and it is one ofthe most important fertilizer in agricultural industry, too. The usage of nitrogen fertilizer couldimprove the quantity and quality of agricultural products，however，it can also causes a lot ofenvironmental and economic problems, for example, the losing nitrogen would flow intowater or the atmosphere, causing eutrophication or global warming. Nowadays, nitrogenpollution has become a serious pollution spread over the world, threatening the wholeecosystem.Glutamate dehydrogenase （GDH） catalyses the formation of glutamate from NH₄⁺andα-ketoglutarate or its reversible reaction, and it plays an important role in the nitrogenmetabolic pathway in vivo. In this paper, all of the fungal protein sequences collected byNCBI and Uniprot were downloaded （about2million sequences）, and then295GDHs from178fungal strains were indentified from the dataset. Apart from that,15GDHs were friendlyprovided by Fujian Agriculture and Forestry University. Further analysis indicated that all ofthe GDHs can divided into two sub-families: one containing GDHs that have a molecularweight about120KDa, using the NADH as co-enzyme and function on catabolizing ofglutamate; another including GDHs that about120KDa, depending on NADPH and are morelikely involved in synthesizing glutamate. The sub-family that have smaller molecular weightshare high similarity with NADPH-dependent GDH of E. coli in structure （primary, secondaryand tertiary structure） and active site, which indicates that GDHs from this sub-family could,just as the GDH from E. coli did, assimilate NH₄⁺efficiently.The GDH from Alternaria brassicicola was chosen for further analysis to confirm thebioinformatic results. A series experiment were performed in vitro and the result indicated thatthe Km value of this enzyme to NH₄⁺,α-ketoglutarate and glutamate were2.144mM、2.690mM and96.772mM, respectively. Besides, the coding sequence of this enzyme wasover-expressed in rice. The transgenic seedlings grown in high nitrogen supplementenvironment showed no different with the wild type plants, while they have enhanced plantheight, dry weight and nitrogen content when nitrogen supplement was deficiency. Thisresults demonstrate that the overexpression of the GDH gene could improve the nitrogenusage efficiency of rice.In conclusion, the small GDHs from fungus possess the proficiency of assimilating NH₄⁺efficiently, they can be used as candidates in genetic engineering to improve the plant nitrogenusage efficiency.