Galloylated catechins in the tea plant are the main phenolic compounds that determine the astringency taste of teas. In our previous study, we found that the content of galloylated catechins was affected by the reaction of synthesis, condensation, and hydrolysis in tea plant [Camellia sinensis (L.) O. Kuntze]. Meanwhile, we found that more than one tannase in tea plant could hydrolyze the gallate ester bond of hydrolytic tannins and galloylated catechins. Up to now, the genes encoding tannase have not been fully clear. In this project, in order to excavate those genes involved in the hydrolysis of gallate ester bond of hydrolytic tannins or condensed tannins, total tannase in the tea plant were firstly isolated and purified by ammonium sulfate precipitation, hydrophobic interaction chromatography (HIC), and gel filtration. The purified proteins were identified by MALDI-TOF-MS/MS, and the candidate genes were screened based on the mass spectrometric datas from tea plant genome and transcriptome databases. The heterogeneous expression of candidate genes in E. coli and purification of recombinant tannase were performed, and biochemical characteristics of the recombinant enzyme were analyzed in vitro. Then, the relationship between the structure and catalytic mechanism of different tannase proteins were analyzed by enzyme kinetics, crystal structure analysis and point mutation technique. Furthermore, the physiological functions of different tannase genes were evaluated by using the established transgenic system, including instantaneous expression of tea plant. Excavation of different tannase genes in the tea plant has an important scientific significance and valuable application to develop tea varieties with low astringency and to screen the excellent germplasm resources from tea plants, grapes and persimmons.

Functional verification of different tannase genes in the tea plant [Camellia sinensis]

256th National Meeting (2018)

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