Ectopic Expression Of A Glutamate Dehydrogenase Gene,EcGDH,from Eurotium Cheralieri Confers Tolerance To Dehydration Stress In Rice

Drought stress is one of the adversities that threaten the normal growth of rice(Oiyza sativa L.),an important crop,and affects its yield.When plants under drought stress,can lead to accumulation of NH4+ in the cells.The excessive NH4+ is toxic to cells.There are two NH4+ assimilation pathways in plants:GS/GOGAT and GDH pathway.However,GDH has a low affinity with NH4+ in higher plants,which makes this pathway not effective for the ammonia assimilation,but the energy consumption of GDH pathway is less than GS/GOGAT.Therefore,to increase the ability of GDH to assimilate NH4+ in plants,GDH can be used to ammoniaify NH4+ to glutamic acid,thereby reducing the detoxification effect of high concentration NH4+ in the cell caused by drought stress,and it is an effective way to resist drought stress.Based on this study,the GDH gene was cloned from Eurotium chevalier and transferred to rice(Oiyza sativa cv.Kitaake),conduct related research about this gene of drought tolerance mechanism in rice.1.The prokaryotic expression of the recombinant protein in vitro,analysis the enzyme activity of the purified EcGDH protein and the GDH in the transgenic lines.The resuLts showed that the activity of ammoniation activity of His-TF-EcGDH was higher than deaminase activity and the activity of NADP(H)-GDH enzyme in transgenic lines was higher than that in the wild type in vivo,and also the ammoniation activity of was higher than deaminase activity under treatment.2.The phenotypes of transgenic lines under normal and simulated drought stress were analyzed and observe the stomatal morphology.Under dehydration stress,the drought resistance of transgenic plants was significantly higher than that in wild-type and the proportion of fully open stomatal was significantly lower than in the wild type.3.Analysis the content of NH4+,glutamic acid,proline and soluble sugar under the normal and dehydration stress in transgenic lines and wild type.Analysis the change trend of proline transportator genes and dehydrate-inducible genes of transgentic lines and wild type in mRNA level by using quantitative PCR under normal conditions and dehydration stress.Under the condition of dehydration stress,the content of NH4+ in transgenic lines was lower than in wild type,and the content of glutamate,proline and soluble sugar was obviously higher than wild type.Quantitative PCR results showed that the gene expression in transgenic lines significantly higher than that in wild type under dehydration stress.4.Due to the oxidative damage associated with drought stress,in order to verify whether the overexpression of EcGDH gene can improve the oxidation resistance in rice,a series of oxidation-related indicators of CAT,POD,SOD and MDA were analyzec.The enzyme activity of CAT,POD and SOD were increased under the simulated drought treatment,and the content in the transgenic lines was higher than that in the wild type,while the content of MDA was lower than that in the wild type.In conclusion,overexpression of EcGDH in rice can increase the activity of GDH enzyme,cause glutamate production from excessive accumulation of NH4+ caused by stress,and reduce-the cytotoxicity caused by high NH4+,thereby increasing the drought resistance of EcGDH-overexpressing rice lines,it lays a theoretical foundation for the search for drought-resistant new rice varieties.