Abstract:In this study, based the homologous sequence of glutamine synthetase (contig48) screened out from young root cDNA library, we designed primers and cloned its full length (named as GS12, GenBank accession number: JQ925873.1) using SMART RACE. The results indicated that: (1) the fulllength cDNA of GS12 is 1 710 bp with an open reading frame (ORF) of 1 071 bp, encoding 356 amino acids with deduced molecular weight of 39.3 kD and theoretical pI value of 5.65. The result of amino acid sequence alignment in NCBI indicated that it has high similarity with the cloned theanine synthetase from Anji white tea. (2) The gene was cloned into prokaryotic expression vector pET32a and pMALc5x, which was further transformed into Escherichia coli Rosetta and BL21 to induce fusion protein with IPTG, respectively. The SDSPAGE results showed that: after induction with IPTG, an insoluble inclusion body would be produced in E. coli Rosetta using the pET32a expression vector, while a soluble protein could be induced in E. coli BL21(DE3)using the pMALc5x expression vector. (3) After the yeast expression vector pYESDEST52CsGS12 was constructed by Gateway technology, the gene was expressed in Saccharomyces cerevisiae (WAT11), and with addition of substrates in medium, the glutamine concentration in strains with pYESGS12 vector transformant was twice as high as that containing the pYES empty vector transformant using UPLCMS analysis. The preliminary result suggested that the GS12 is capable of synthesizing glutamine instead of theanine.