Colning, Heterologous Expression Of Theanine Synthetase Of Tea Plants (Camellia Sinensis) And Its Regulation By Nitric Oxide

Tea is the most consumed non-alcoholic beverages in the world. L-theanine is thecharacteristic and non-protein amino acid in tea plants (Camellia sinensis), which notonly determines the flavor of tea but also has a wide range of pharmacologicalfunctions. Theanine synthetase is a key enzyme in the theanine synthesis, and plays akey role in nitrogen metabolism in tea plants. Previous repots showed that theaninesynthetase gene sequence is highly homologous with glutamine synthetase genesequences. Further studies are needed for our better understanding theaninemetabolism and the mechanism of nitrogen assimilation and transport in tea plants. Inthis study, we cloned theanine synthetase gene (DD410895) and glutamine synthetasegene (AB115184) from tea by RT-PCR, and verified its function through theprokaryotic and eukaryotic expression, and prepared polyclonal antibody throughpurification prokaryotic expression protein. On this basis, we used bioinformaticstools to analyze the structure and function of these proteins, which deduced by TS andGS gene sequences, and using the method of phylogenetic tree to analysis theirlocalization in the GS gene family. Moreover, we analyzed the expression of TS geneand theanine accumulation in tea seedling treated with different NO concentrations,by using the methods of HPLC and Western Blotting techology，respectively. Hereare the detailed results:Total RNA of tea roots and leaves were extracted, then theanine synthetase gene(DD410895) and glutamine synthetase gene (AB115184) were cloned by RT-PCR.The length of obtained TS gene was1071bp, which had three amino acid residuesdifferent from the known amino acid sequences of TS gene in tea, and high homologywith GS genes in plants. The obtained GS gene had the only one amino acid differentfrom the known amino acid sequences of GS gene. The two genes were expression inE.coli, and soluble proteins were obtained. Specificities of the enzyme function werefurther verified. These results show that the expressed protein of TS catalyzestheanine synthesis using glutamine and ethylamine hydrochloride, rather thanglutamic acid and ethylamine hydrochloride. Tea TS has no catalytic activity of GS.Theanine，the product of enzymatic reaction, was identified by HPLC and ESI-MS.GS protein has a catalytic activity for amino transportation, which is much greater than the activity of bacteria GS.Plant binary expression vectors pGREEN-35S-TS/GS, containing TS or GS cDNAsfused with GFP, were constructed and introduced into Agrobacterium, and furthertransferred into the Arabidopsis by the floral dip method. The transgenic Arabidopsiscontaining TS and GS genes was screened by herbicide Basta. The positive plantswere confirmed by PCR. The fusion protein was observed mainly in the cytoplasmin the root tip cells of the transgenic Arabidopsis by using laser scanning confocalmicroscope.The Expasy software packages and some online analysis tools such as CBS,Swiss-Model and InterProScan were used to analyse and predict the physical andchemical properties and the protein structure of TS and GS gene. The results showthat TS theoretical isoelectric point (PI) was5.52, protein molecular weight (MW) is39306.3Da, GS theoretical isoelectric point (PI) is6.13, and protein molecular weight(MW) is39240.3Da. Both of them are soluble, strong hydrophilic proteins, whichhave no transmembrane structure. Secondary structure prediction indicates that theyare all mixed type proteins and little difference between the protein structures. Aminoacid sequence and structural analysis showed that both TS and GS protein includes aGS beta-Grasp domain and a GS catalytic domain, these functional fragments areconserved in glutamine synthetase genes of many plant species and all belong toGln-synt domains. Protein subcellular localization and functional analyses showedthat, TS and GS proteins were localized in the cytoplasm, which have high probabilityto executive some function such as transcription, transcription regulation and signaltransduction. Homology modeling software SWISS-PdbView was used to predictthree-dimensional structure of the TS gene. Moreover, the metapocket software wasused to analyze TS protein active sites through three-dimensional structure of maizeGS protein as a template (No.:2d3a). Three binding sites in the enzyme werepredicted, roughly equivalent to its location with GS, but its sites which couldcombinate amino acid have a slight difference, which is probably the reason for thedifference of the catalytic reaction between the TS and GS. Using Mega4.1software,the phylogenetic tree was built, which indicates that the TS gene belonging to GSfamily members and GS protein of Camellia sinensis can be classified into threecategories.High-performance liquid chromatography (HPLC) detector results revealed that nitrogen monoxide (NO) induced the rapid accumulation of L-theanine in the tea rootrather than in the leaves, and the expression of L-theanine synthetase was bothenhanced in both the roots and leaves. HPLC results also revealed the presence of NOinduced the accumulation of catechins as ester-like compounds in the leaves.Application of NO scavengers and pretreatment with NO-metabolism inhibitorsreduced NO-induced L-theanine accumulation, but no significant effect on catechinsynthesis. These results indicated that L-theanine and catechin metabolism areregulated by different mechanisms in young tea seedlings.