Isolation Of Cytosolic Glutamine Synthetase Genes CsGs1s And Their Experssion In Tea Plant(Camellia Sinensis L.) Under Different Nitrogen Forms

As the tea plant is a perennial economic crop with leaves,adequate nitrogen nutrition plays a vital role in its growth and development,physiological and biochemical metabolic processes and the formation of tea quality.However,The uptake and utilization of nitrogen by tea plant is limited,and the tea plant prefers ammonium nitrogen（NH₄⁺-N）,there are differences in the utilization efficiency of different nitrogen sources.Therefore,in general tea garden management,in order to ensure economic benefits,the excessive application of nitrogen fertilizer is more common.But this will definitely lead to waste of resources and environmental pollution.Glutamine synthetase（GS;EC6.3.1.2）is one of the most important catalytic enzymes in nitrogen assimilation process in higher plants,and it is also closely related to plant nitrogen uptake,assimilation and utilization efficiency（Nitrogen use efficiency,NUE）.More importantly,the gene members of different GS enzymes have different functions in plant nitrogen assimilation.This study is based on Camellia sinensis cv.‘Longjing43’as the reserch object.The full-length cDNA of 3 CsGS1 genes involved in nitrogen assimilation was cloned,the subcellular localization and the protease activity of expression products of GS1s were studied,and the expression patterns of 3CsGS1 genes in different nitrogen sources and their tissue specificity were studied,which was to reveal its specific functional role in the nitrogen metabolism of tea plant.The main findings were as follows:1.We cloned 3 cytoplasmic glutamine synthetase gene CsGS1.1,CsGS1.2 and CsGS1.3.CsGS1s encoded proteins all have the conservative structure domain-gln-synt_c of GS family through bioinformatics analysis.All these three genes CsGS1s encoded hydrophilic and non-secreted proteins which localized in the cytoplasm without transmembrane structure.Through blast analysis and evolutionary tree construction,it is shown that CsGS1.1 has the closest relationship with grape GS1.1,while CsGS1.2 and CsGS1.3 have the closest relationship with poplar GS1,and CsGS1s is closely related to Arabidopsis thaliana1;5、Brassica napus1;5、Hordeum vulgare1;3、Oryza Sativa1;3 and Zea mays1;3.2.The encoding regions of the cloned 3 CsGS1s genes were fused with the GFP gene of the localization report gene,and the expression products of Cs GS1s were precisely subcellular located.The results showed that the CsGS1.1 was located in the cell membrane,cytoplasm and nucleus,and the CsGS1.2 was located in the cell membrane and the cytoplasm,and the CsGS1.3 was located in the cell membrane and the cytoplasm.3.Prokaryotic expression was performed on 3 CsGS1s coding regions,and the corresponding recombinant proteins were purified.SDS-PAGE electrophoresis results showed that the protein CsGS1.1,CsGS1.2 and CsGS1.3 were about 42,42 and 58 KD respectively.The catalytic characteristics of glutamine synthetase of recombinant proteins showed that 3 recombinant CsGS1s proteins all had GS enzyme catalytic activity.4.The expression of CsGS1s gene is temporal-spatial specificity.Analysis of CsGS1s tissue specificity in tea plant showed that CsGS1s were expressed in root,stem and leaf,interestingly,the transcription level of CsGS1.2 was rather low.The expression level of CsGS1.1/CsGS1.3 was highest in root while CsGS1.2 was mainly expressed in the leaf.Furthermore,the expression patterns of CsGS1s in response to different nitrogen forms in tea plant were detected using quantitative real-time PCR analysis.In leaves,the transcription of CsGS1.1 was just induced by nitrate until the 48 h treatment time point during the whole nitrogen treatment time course.Contrary,the expression of CsGS1.2/CsGS1.3 was highly induced by ammonium and with a slight induction by nitrate.In roots,the response of CsGS1s to ammonium was rather higher than that of nitrate,especially for CsGS1.1/CsGS1.2.