Study On The Synthesis And Degradation Of Bitter And Astringent Polyphenols In The Tea Plant(Camellia Sinensis)

Tea is rich in polyphenols,including flavonoids,catechins,anthocyanins and phenolic acids,which endow tea with unique taste and quality characteristics.Studies have reported that esterfied catechins are the major contributions to astringency and bitterness of tea drinks,and flavonol glycosides with very low astringent thresholds impart a silky,dry and astringent sensation upon consumption.High accumulation level of esterfied catechins and flavonol glycosides in tea plants will greatly degradated the quality of tea beverages.This paper focuses on several key genes involved in the synthesis and degradation of bitter and astringent polyphenols.The results are as follows.1,In this paper,we screened and cloned one gene CsUGT75L12,three genes CsRHMa,CsRHMb and CSRHMc encoding the biosynthesis of UDP-rhamnose,and one gene CsUGT79C3 encoding the biosynthesis of flavonoid 7-O-neohesperidin from the transcriptomes of different tissues and organs of tea plants by means of bioinformatics.The functions of these genes were verified by prokaryotic expression,enzymatic characterization and mass spectrometry.The results of gene expression,protein localization and product accumulation patterns analysises showed that the synthesis of the 7-O-neohesperidin in tea plant was catalyzed by the combined action of flavonoid 7-O-glucosyltransferase coded by CsUGT75L12,UDP-rhamnose synthase coded by CsRHMs and flavonoid 7-O-glucose(1→2)-O-rhamnose transferase coded by CsUGT79C3.This study not only fill the gap of biosynthetic molecular mechanism of flavonoid 7-O-glucoside and UDP-rhamnose in tea plants,but also provide molecular basis for breeding new cultivars of tea and improving the tea quality.2,In this paper,the tannin acyl hydrolase(TAH)was isolated and identified from tea plants by means of protein purification,protein sequencing and enzymology,which was named as CsTA.Prokaryotic and eukaryotic expression techniques were applied to express CsTA fusion protein.enzyme activity analysis In vitro showed that the both CsTA fusion proteins had tannin acylhydrolase activity.A family of plant tannases that are evolutionarily independent of carboxylesterase was screened from the tannin-rich species using the CsTA protein as homologous sequence by using means of bioinformatics.Similarly,the fusion proteins of VvTA in grape,FaTA in strawberry,DkTA in persimmon,CcTA in citrus and JrTA in walnut were expressed by prokaryotic expression technique.The results of enzyme activity analysis in vitro showed that the fusion proteins all showed tannin acylhydrolase activity.Quantitative analysis showed that the expression of CsTA gene was positively correlated with the hydrolysis of esterified catechins in tea plants.Overexpression of FaTA gene and RNAi interference assays showed that the FaTA gene was involved in the synthesis of hydrolyzed tannins in strawberry.In conclusion,we isolated and identified a class of plant tannases for the first time.