Molecular Cloning, Expression Of Theanine Synthetase Gene And Determination Of Theanine In Camellia Oleifera

HPLC and LC-TOF/MS were applied to determine theanine in the roots and leaves in the young and mature Dabieshan wild Camellia oleifera. Meanwhile molecular biology methods were used to clone theanine synthetase (TS) gene of C. oleifera and then analyze the gene in bioinformatics. Thereafter, prokaryotic expression about TS of C. oleifera were carried out. The TS in young and mature roots, leaves were also studied by qPCR. The results revealed that the theanine content of roots in young C. oleifera plants was 0.08 mg.g-1 (fresh weight). However, theanine was not detected in leaves of young plants. It was also found there is no theanine in the roots and leaves in mature C. oleifera. One TS ORF (1071 bp) was acquired from the roots in young C. oleifera. TⅠⅠⅠⅠhe homology of the cloned TS with glutamine synthetase gene(GS, AB 117934) and TS (DD410896) were up to 98%. Moreover, the cloned TS showed a high similarity about 99% to GS (AB117934) and TS (DD410896) from C. sinensis at the protein levels. Bioinformatic analysis showed that there were 20 phosphorylation sites in the polypeptide chain after. Bioinformatics prediction demonstated that the protein contained hydrophilic and winded helix domain. However, there was neither signal peptide nor transmembrane in the sequences, and the protein was non-secreted which functioned in the cell. Otherwise, the prokaryotic expression system for TS was constructed. TS was cloned into plasmid vector PGEX and transformed into E.coli Rosetta strain. The recombinant was reduced by IPTG with the concentration of 1 mM at 37 ℃ for 4-5 h. The obtained protein with molecular weight of 39.5 kD was in correspondence with the initial prediction. Expression of the TS fusion protein in soluble part was low and mainly existed as inclusion body. The expression of two pairs of primes TS1, TS2 in roots was much higher than in leaves of the same growth C. oleifera. Moreover, TS1, TS2 expression in young roots were significantly higher than roots and leaves in other period of C. oleifera. On the whole, TS transcription level in various periods and various position of C. oleifera were different.Results in this paper will provide theoretical basis and new approach for study of the synthesis, metabolic pathways and metabolic mechanism of theanine. Furthermore, it beneficial to develop economical value of C. oleifera.