| Tea contains polyphenolic compounds,which include flavanols,flavandiols,flavonoids,and phenolic acids.The main contents of the polyphenols in tea plants are flavanols,commonly known as catechins,their total concentration is around65%-80% of tea polyphenolic compounds.Catechins play a significant role in tea quality and efficacy,and have antioxidant,antiallergy,antitumor and antiaging activities.Flavonoid 3?5?-hydroxylase,which belongs to the cytochrome P450 family,is the only enzyme catalyzing the 5? position hydroxylation of the B-ring.Flavonoid3?5?-hydroxylase(F3?5?H)is the key enzyme which can synthesize delphinidin-3-glucoside in anthocyanin biosynthesis,so called “blue gene”.In this study,buds of cv.Shuchazhao as the experimental materials were peeled into two parts,shoot tip(ST)and unexpanding young leaf(YL),to investigate the accumulation patterns of catechins by high performance liquid chromatography(HPLC),dimethylamino-cinnamaldehyde(DMACA)staning,real time fluorescence quantitative PCR(q RT-PCR)and in situ hybridization technologies.In this paper,a new F3?5?H gene(Cs F3'5'H2)was screened out from the tea purple buds transcriptome,and the full-length c DNA of Cs F3?5?H2 was cloned,The Cs F3?5?H2gene's tissue-specific expression and the expression under abiotic stress were performed by q RT-PCR.Then eukaryotic expression was constructed to verify the gene function in vitro;by replacing signal peptide of Cs F3'5'H2,we constructed Cz F3'5'H2.The study on the gene expression profile of catechins in the tender organ will lay the foundation for the research of key regulators in the biosynthetic pathway of catechins.The main research results are as follows:1.HPLC analysis showed the total amount of catechins was significantly higher in the YL than the amount of catechins in the ST.Compared with ST,the concentration of gallated catechins and non-gallated catechins were increased both in Y.The concentration of gallated catechins EGCG and ECG in YL,increased by74%and 71.8% than that in ST,and the non-gallated catechins GC,EGC,C and EC respectively increased by 4.0,1.3,1.0 and 1.2 times.2.Through DMACA staining,the histological localization of catechins in tea buds were studied.The results showed that catechins accumulated in mesophyll cell(including palisade tissue and spongy tissue)and bud axis,in the young leaf epidermis and leaf primordium were not observed staning.3.The expression patterns of genes involved in the flavonoid pathway were analyzed by q RT-PCR.The results showed that expression levels of the catechins metabolism related genes(PAL?C4H1?C4H2?CHS2?ANR1?DFR1?DFR1 and F3?5?H1)in YL were significantly higher than that of ST.4.The further detection of gene expression with situ hybridization indicated that ANR1,DFR1,DFR2 and LAR were mainly expressed in the leaf primordial and the young leaf of tea buds.Nevertheless,hybridization signals of ANR2,F3?5?H1 and SCPL4 were very weak in tea buds.5.Quantitative RT-PCR analysis showed that the Cs F3?5?H2 gene expressed in bud,leaf,stem and root,and it had the highest expression in the first leaf.Sucrose and abscisic acid could induce upregulation of Cs F3?5?H2 expression.6.The recombinant plasmids of p YES-dest52-Cs F3?5?H2 and p YES-dest52-Cz F3?5?H2 were constructed and transformed into expression of host Saccharomyces cerevisiae WAT11,which achieving Cs F3?5?H2 and Cz F3?5?H2expressed in WAT11.And enzyme activity on different substrates had been detected.The results showed that Cs F3?5?H2 can catalyze dihydroquercetin,while Cz F3?5?H2can catalyze dihydroquercetin and dihydrokaempferol. |
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