Overexpression Of An NADP(H)-dependent Glutamate Dehydrogenase Gene,TrGDH,from Trichurus Improves Nitrogen Assimilation And Yield Of Rice

Although glutamate dehydrogenase?GDH?is widely present in plants,but has low affinity for NH₄⁺,as glutamate dehydrogenases?GDHs?of microorganism usually have higher affinity for NH₄⁺than those of higher plants,it is expected that ectopic expression of these GDHs can improve nitrogen assimilation in higher plants.Based on recent studies,the target gene of this study,was cloned from Trichurus and transferred into rice?Oryza sativa cv.kitaake?to analysis its effect on nitrogen utilization and yield of rice.?1?A novel NADP?H?-GDH gene?TrGDH?was isolated from a fungus Trichurus,the total length of the gene was 1359 bp,encoding 452 amino acids.The phylogenetic tree analysis of TrGDH and other 33 GDHs from different sources showed that TrGDH was on the same branch of the phylogenetic tree as Chaetomium globosum and Neurospora crassa,but on different branches with higher plants such as rice and rabidopsis thaliana,with low affinity.?2?The prokaryotic expression of the Tr GDH recombinant protein,the recombinant protein was 104 kD.Investigation of kinetic properties in vitro showed that Tr GDH exhibited higher affinity for NH₄⁺?K_m=1.48±0.11 mM?compared to rice GDH?Os GDH4?,and the catalytic reaction was in the direction of generating glutamate.?3?We constructed overexpression vector of TrGDH,named pCAMBIA1301GW-Tr GDH,and transferred into ricemediated by agrobacterium tumefaciens.Measurements of NH₄⁺assimilation rate demonstrated that NADP?H?-GDH activities of Ubi::TrGDH transgenic lines were significantly higher than control.Through qRT-PCR analysis,it was found that TrGDH transfected into rice competitively inhibited the expression of rice endogenous GDHs to some extent.?4?The nitrogen stress hydroponics experiment revealed that,all fresh weights,dry weights and nitrogen contents were significantly increased in Ubi::TrGDH transgenic lines compared to the control.Field trial further demonstrated that all the number of effective panicles,1,000-grain weight and grain weight per plant of transgenic lines were significantly higher than control,especially at low-nitrogen levels.In addition,the determination of protein in seeds of Ubi::TrGDH transgenic plants showed that the content of glutenin and prolamine in the transgenic plants was markedly increased compared with control,indicating that the quality was improved.To sum up,these results sufficiently confirm that overexpression of TrGDH in rice can improve growth status and grain weight per plant by enhancing nitrogen assimilation.Thus,this study provides a theoretical and experimental basis for TrGDHto become a promising candidate gene for enhancing crop yield by genetic engineering.