Study On The Accumulation Of Flavonol Glycosides In Tea Plants [Camellia Sinensis(L.)O.Kuntze] And The Function Identificition Of CsFLS Gene

Phenolic compounds are major components in the flavour of tea liquor,inside flavonol glycosides play an important role in the astringent taste of tea infusion.The flavonol mostly accumulated as flavonol glycoside in the tea plant,and is directly synthesized by flavonol synthase(FLS)from dihydroflavonol.Therefore,the accumulations of flavonol are closely related with flavonol synthase.In this paper,the flavonol glycoside in the tea plant was identified and quantified.In order to further study the accumulation of flavonol glycoside in the tea plant,the function of the flavonol synthase was also studied.The results of the study are as follows:1 The identification and quantification of flavonol glycoside in tea plantA total of 15 flavonol glycosides in the tea plant was identified by HPLC-TOF-MS,and quantified by an established method based on multiple reaction monitoring(MRM)by ultra-high performance liquid chromatography(UPLC)coupled with a triple quadrupole mass spectrometer(QQQ-MS)instrument.Besides,the standards of six flavonol glycosides were used to assess the effectiveness of the method.The results showed that the obtained standard curves of six flavonol glycosides all had good linear correlations(R2> 0.99)in a certain concentration ranges.The values of LOD and LOQ were less than 0.01 and 0.03 ng,respectively;the values of recovery varied from 90% to 105%;the values of intra-day RSD of retention time and signal intensity(peak area)were less than 0.15% and 3.9%,respectively;and the values of inter-day RSD of retention time and signal intensity(peak area)were less than 0.26% and 4%,respectively.Thus,it suggested that this method had a good sensitivity,precision and reproducibility.The content of flavonol glycosides were quantified by the MRM mode of UPLC-QQQ-MS/MS.In the four tea cultivars studied,most flavonol mono-,di-and tri-glycoside were found mostly to accumulated in the young leaf,a small amount in the bud,mature leaf,and young stem;inside the content of quercetin 3-O-rutinoside was also high in the mature leaf and young stem.Moreover,six flavonol glycosides were absolutely quantified by the corresponding authentic standard.The results showed that they were abundant in young leaves which were consistent with the results of the relative quantification.Besides,the results also showed the content of them were extremely low in the old stem and root.2 Function analysis of flavonol synthase gene in the tea plantIn this paper,three FLS(flavonol synthase gene)genes were isolated from tea plants,besides the two FLS gene which had been researched previously,here we cloned a new FLS gene in tea plant and named as FLS3.QRT-PCR assay was performed to analysis the relative transcript level of three FLS genes in different developmental stage leaves and organs,as well as under the different treatment conditions.The three FLS genes were detected in bud,first leaf,second leaf,mature leaf and young stem.CsFLS1 gene showed a higher transcript level in bud compared to the other organs.In contrast,CsFLS2 and CsFLS3 gene showed a higher expression level in second leaf.In addition,the expression level of three FLS could be induced under sucrose and ABA treatments,but not be induced by the other treatments.In order to analysis the function of three CsFLS genes,the ORF(open read frame)of these three genes were cloned into pRSFDuet-1 expression vector with a His-Tag in N-terminal.The three CsFLS genes were expressed in E-coli BL21 and the recombinant proteins were purified by using a Ni affinity system.The vitro activity of the recombinant CsFLS proteins was detected by HPLC.The recombinant CsFLSs proteins can catalyze K,Q,and M from substrates of DHK,DHQ,and DHM,respectively.The recombinant CsFLS2 protein revealed a higher activities to substrate DHK with a Km of 22.9?M,but lower activities toward DHQ and DHM with a Km of 80?M and 65.55?M respectively.In addition,the ORF(open read frame)of CsFLS2 gene was cloned into PCB-2004 expression vector and transformed into tobacco plants.It is interesting that the flowers of CsFLS transgenic tobacco plants had a lighter color phenotype compared to wild-type tobacco plants.The UPLC-MS analysis results showed that the CsFLS transgenic tobacco flowers accumulated more flavonol glycosides but less anthocyanin.The subcellular localization observation indicated that recombinant CsFLS2 protein was mainly distributed in cytoplasm and cytoblast in Nicotiana benthamiana plants.