Cloning And Prokaryotic Expression Of Three Endogenous Glycosidases Involved In Tea Aroma Formations From Camellia Sinensis

Tea (Camellia sinensis) is one of the most important economic plants in China, andtea aroma is a critical index of tea quality. Glycosides are the main form precursors of thevolatiles such as glycosides of monoterpene alcohols and aromatic alcohols in tea leaves.Most of the glycosidic aroma precursors are primeverosides and glucosides. Glycosides area kind of secondary metabolites in the leaves of Camellia sinensis during growth. Enzymesresponsible for biosynthesis and hydrolysis of glycosides mainly includeβ-primeverosidase, β-glucosidaseⅠand β-glucosidaseⅡ.The volatile aglycones released from glycosides are not only the material base of teaaroma, but also participate in the tea plant defense reaction against disease and pests. Toinvestigate the cloning and expression of the genes encoding endogenous glycosidases,will provide valuable insights into the regulation of gene expression related with tea aromabiosynthesis. To identify factors affecting tea aroma and the mechanisms relationships ofthe endogenous glycosidases, will help to propose the prospect of using exogenousinducers to regulate tea aroma.The primers were designed and synthesized according to full-length sequence ofβ-primeverosidase (EC3.2.1.149) and two kinds of β-glucosidases from tea plants(Camellia sinensis) which we have reported the purification before. The ORF (OpenReading Frame) was amplified by RT-PCR, which was subsequently cloned into pMD18-Tvector for sequencing analysis. The results of sequencing analysis showed that thenucleotide sequences had high identity (up to99%) with the original sequence.Bioinformatics analysis showed that the full length Pri was1729bp, with1524bp ORFwhich encoding507amino acids; the full length GluⅠ was2337bp, with1896bp ORFwhich encoding632amino acids; the full length GluⅡ was2215bp, with1887bp ORFwhich encoding629amino acids. And the protein molecular weight were about57.04kD、69.15kD and68.87kD, respectively. Evolutionary analysis showed that three kinds ofendogenous glycosidases from the tea plant had the close relationship with β-glucosidase.Moreover, both GluⅠ and GluⅡ had high identity with β-glucosidase from Vitis vinifera.However, Pri was close to β-glucosidase from Camellia sinensis, and far from GluⅠandGluⅡ.Three kinds of endogenous glycosidases had been expressed in prokaryoticexperimental system. β-primeverosidase, β-glucosidaseⅠand β-glucosidaseⅡ were clonedinto pET-32a (+) expression vector, and the recombinant plasmid were transformed to Rosetta (DE3) pLysS, respectively. The impacts of different induction time andtemperature on the expression were investigated and the enzymatic activities weredetermined.The expression results revealed that all these three kinds of endogenous glycosidasesgene can be highly expressed in E.coli Rosetta (DE3) pLysS after induced by IPTG for5hat37℃. However, these three recombinant proteins were in the form of inclusion body,and the protein molecular weights were about56kD,70kD and68kD, respectively.