Functional Analysis Of Overexpressing Glutamine Synthetase, Nitrate Transporter And Ammonium Transporter 1 Genes For Nitrogen Assimilation In Rice

At present,as the society's industrialization process increased,the cultivated land in China is decreasing day by day.In order to satisfy people's demand for food,a mount of chemical fertilizers especially nitrogen fertilizer were applied,and it becomes to be the main method to improve grain yield during agricultural production in our country. However,with the substantial inputs of nitrogen fertilizer,the efficiency decreased and accompanied by large scale pollution of water resources in our enviroment.Rice is one of the most important crops in China and even the whole earth,together with the fast development of molecular biology techniques in recent years,the research work about genes involved in nitrogen metabolism in flee,and breeding new rice variety of high efficiency of nitrogen absorption and untilization by transformation method,will play a very important role in rice production and even the entire agricultural development in our country.In this study,genes including glutamine synthetase(GS),nitrate transporter(NRT) and ammonium transporter(AMT) involved in nitrogen metabolism were over-expressed in rice plants,the phenotype and metabolic level were analysed in transgenic plants by molecular biology and physiology methods,in order to obtain the transgenic rice plant which exhibited high efficiency of nitrogen absorption and untilization.The main results are as follows:1.The sequence information of genes involved in nitrogen metabolism,including GS1;1,GS1;2,GS1;3,GS2,glnA,NRT2,AMT1;1,AMT1;2 and AMT1;3,were obtained from NCBI database using bioinformatic methods.And the full length coding sequences were isolated by PCR amplification or cloned from cDNA library.Based on the pCAMBIA1301S vector which contained 35S promoter,nine constructs overexpressed GS1;1,GS1;2,GS1;3,GS2,glnA,NRT2,AMT1;1,AMT1;2 and AMT1;3 respectively, were transformed into rice plants by an Agrobacterium tumefaciens-mediated transformation method.The copy numbers and expression level of transformed genes were also checked by Southern and Northern blot respectively.2.The phenotype and biomass of GS1;1-,GS1;2- and glnA-overexpressed plants exhibited no significant changes when compared with wild type plants.The results of biochemical and physiological testing showed a higher metabolic level in the transgenic plants compared with wild type plants,including GS activities,soluble protein content, free nitrate and ammonium content,free amino acids,total nitrogen and total amino acids content.However,the decreased plant yield and seed amino acids content were observed in the transgenic plants.The results of abiotic stress indicated GS1;2-overexpressed plants showed a significantly high resistance to Basta,and were highly sensitive to high concentration of NaC1,cold and drought stress conditions compared to the wild-type plants.While the transgenic plants overexpressing glnA showed sensitive to high concentration of NaCl,but not to Basta,cold and drought stress conditions.However,the transgenic plants overexpressing GS1;1 did not show any changes to the abiotic stress condition mentioned above.3.The GS2-overexpressed plants displayed a phenotype of yellow leaves,less tillers, decreased biomass and yield in the T₁ generation.Although the transgenic plants were small and leaves were yellow,they still can finish the whole life with few seeds.The results of biochemical and physiological testing showed a lower metabolic level in the transgenic plants compared with wild type plants,including GS activities,soluble protein content and free ammonium content.As the total free amino acids were not significantly changed,however,the individual amino acids changed significantly,especially the Asx and Glx content.The phenotype of yellow leaves in GS2 transgenic plants was not based on the genotypes,it caused by the GS2 co-suppression.The yellow leaves only exhibited after 2-leaf stage and in the young leaves at the top of the mature plants,while the two old leaves kept green at the bottom.This yellow leaves could be returned green aider the exogenous glutamine added to the nutrient solution.4.The phenotype and biomass of GS1;3-,NRT2- and AMT1;2-overexpressed plants exhibited no significant changes when compared with wild type plants.However,a T-DNA insertional mutant was found amoung the NRT2-overexpressed families which exhibited the phenotype of rolled root,while there was no changes in the shoot and no biomass changed compared with wild type plants.The AMT1;3-overexpressed plants exhibited small plants,decreased plant height and fresh weight when compared with wild type plants.The results of free ammonium content testing showed that,there was a reduction of free ammonium content both in root and shoot.The expression level of GS genes in transgenic plants showed no significant changes compared with wild type plants by Northern blot analysis.These results mentioned above indicated that,a higher nitrogen absorption,utilization and metabolic level was really showed in transgenic plants overexpressing GS1;1,GS1;2 and glnA,although there were no better phenotypes,such as increased biomass or yield, when compared with wild type plants.And there are still amount of work to be continued to identify the impact of nitrogen related genes tansformation on nitrogen metabolism, especially the work about transgenic plants overexpressing GS1;3,NRT2,AMT1;1, AMT1;2 and AMT1;3.