The Mechanism Analysis Of Rice 14-3-3 Protein Regulating Plant Nitrogen Metabolism

The rice production of China is facing increasing nitrogen fertilizer and the phenomenon of nitrogen fertilizer utilization continuing low,leading to farmland soil acidification,groundwater pollution and rivers and lakes eutrophication and a series of environmental pollution problems.Therefore,how to improve the utilization rate of nitrogen fertilizer in rice is an important scientific problem in the current rice breeding and cultivation.In recent years,with advanced genetic engineering technology,the efficiency of crop uptake and utilization of nitrogen was improved by molecular modification method,had becomed an important research direction for improvement of high efficiency of nitrogen nutrition narieties in Rice.In this study,the rice 14-3-3 protein was the main research object,by means of protein interaction and reverse genetics research methods,the mechanism of 14-3-3 protein involvement in plant nitrogen metabolism under low nitrogen stress was elucidated,the results can provide a theoretical basis for the next step in improving the nitrogen absorption and utilization of rice.The main results are as follows:1.Through the bioinformatics analysis of 14-3-3 family of rice,and using RT-PCR method,the expression trend of OsGF14e members were found in the 14-3-3 family of rice was a negative correlation between the expression of three key proteins Nitric acid reductase(NR)、glutamine synthetase(GS)and glutamate synthase(GOGAT)in the nitrogen metabolism pathway under low nitrogen stress.Furthermore,118 target proteins interacting with GF14e were identified by GST-Pull Down technique in rice leaves,NR、GS、GOGAT these three key metabolites of nitrogen metabolism were identified.At the same time,selected the NR protein,by the method of pull down combined with Western blot,it is confirmed that GF14e does have a direct interaction with nitrogen metabolism-related proteins.In addition,the results of protein interaction also showed that GF14e protein was associated with photosynthetic reaction or chlorophyll-related protein such as light chlorophyll protein complex(LHCP),light system Ⅱ reaction center subunit(PsbS);with carbohydrate metabolic pathway-related proteins such as(AGPS)and sucrase synthase 1(SUS1)and other proteins in signal transduction,cell mechanism and growth,protein synthesis and amino acid synthesis,indicating that the 14-3-3 protein is extensively involved in various key metabolic processes of plants.2.Transcriptional studies had showned that,the expression trend of GF14e gene was opposite to that of the key enzyme of nitrogen metabolism under low nitrogen stress,this study founded that with the increase of the amount of exogenous OsGF14-e purified protein,the activities of NR,GS and GOGAT were decreased by complementary experiments in vitro,suggesting that the GF14e was a negative relationship of regulation between the key enzyme of nitrogen metabolism.In this study,we constructed the interference plant and overexpression plant of GF14e,in OsGF14-e-interfering(ER)plants,the activity of the three key enzymes of nitrogen metabolism was significantly higher than that of wild type and overexpressing plants,it confirmed that the relationship between GF14e and NR、GS and GOGAT were negatively regulated.3.In order to investigate the effect of OsGF14-e protein on plant nitrogen metabolism and growth,in this study,the agronomic traits and yield indexes of OsGF14-e transgenic plants were investigated by setting three different nitrogen application rates.The results showed that the SPAD value,photosynthesis intensity,tillering and dry matter weight of OsGF14-e-interfering plants were significantly higher than those of wild type plants and overexpressing(EO)plants under different nitrogen application rates;at the same time,OsGF14-e disturbed plants had higher N uptake and nitrogen content than wild-type and overexpressed plants,and had higher nitrogen uptake and utilization rates(nitrogen uptake rates were their 1.23 times and 1.21 times),nitrogen agronomic utilization,nitrogen harvest index and nitrogen transport rate,indicating that OsGF14-e(ER)plants had a more significant nitrogen absorption and utilization capacity.In conclusion,there was a significant negative correlation between OsGF14-e protein in rice 14-3-3 protein family and nitrogen metabolism key enzymes(NR,GS,GOGAT),specifically reducing the expression of OsGF14-e in plants,under low nitrogen treatment,the activity of enzymes related to nitrogen metabolism could be improved effectively,improved the nitrogen use efficiency of rice plants,and promoted the growth of rice plants,the yield of rice could be ensured in the case of appropriate reduction of nitrogen fertilizer application.