Sparation&Purification Of Methylated EGCG And Cloning&Expression Of Related Genes In Camellia Sinensis

EGCG is a kind of secondary metabolite which has important physiological function in tea.(-)-epigallocatechin-3-O(3-O-methyl) gallate(EGCG3"Me) and (-)-epigallocatechin-3-O(4-O-methyl) gal late (EGCG4"Me) are O-methylated derivatives of EGCG. The experimental results show that they have potent anti-allergic effect, especially anti-pollen hypersensitivity, Japanese industrial circles have pay more attention to their advantages. Allergy has been defined as a disease of excessive immune activity, the morbidity of allergy is very high in some countries and increases with the development of civilization, for example in Japan, the morbidity of allergy is estimated to be more than30%. Now people often use chemosynthetic medicine to heal allergic disease, they have misgivings about the use of anti-allergic medicine because of mounting medical expenses and side effects, but researchers have not found other pure plant drugs whose function of anti-allergy, especially anti-pollen allergy are more than EGCG3"Me and EGCG4"Me in tea leaves. Health product and international medical industries think it is highly possible for methylated EGCG to be developed into a single variety resources and consequently become to be a new economic growth point for catechins which will bring about great wealth to the society. In this study, HPLC method was established for determination of methylated EGCG from tea leaves, methylated EGCG was isolated and purified, genes which related to the property of methylated EGCG were cloned and their functions were analyzed preliminarily. The results may show theoretical and technical support for regulating biosynthesis of metylated EGCG. The main results were as follows:A analytic method was developed to determine eight kinds of catechins, three kinds of purine alkaloids and gallic acid in tea by HPLC-PDA. The optimum condition was as follows:Welchrom Cis column; mobile phase:phosphate buffer-acetonitrile; flow rate:1.0mL/min; wavelength:278nm; column temperature:30℃. This method showed good linearity among injection and peak area. The HPLC method I have developed offers a kind of modern analytical method for screening tea germplasm resource and monitoring the course of separation and purification for methylated EGCG in tea.A separation method was established to separate and purify EGCG4"Me and EGCG3"Me in tea leaves by using column chromatography and preparative high performance liquid chromatography. Crude extracts were obtained from tea leaves by using water and ethyl acetate as extraction solvent. Then the concentracted crude extract was separated on a HP-20column by using different concentration of ethyl alcohol as eluant. The Fr-1fraction was obtained from the eluate of80%ethyl ethanol, then concentrated and subjected to a preparative high performance liquid chromatography for isolation of target components by using water-acetonitrile as mobile phase. Two monomeric compounds were acquired and identified. They were EGCG4"Me and EGCG3"Me.The purity of them was more than98%.A chemical method was established to synthesize methylated EGCG by using dimethyl sulfate as methylated reagent. Methylated EGCG product was separated by using preparative high performance liquid chromatography and supercritical fluid chromatography.Three kinds of monomeric compounds were acquired and identified. They were EGCG4"Me,3’,4"-di-O-methyl-EGCG and4’,4"-di-O-methyl-EGCG, the main product is EGCG4"Me. The purity of them was more than98%.Methylated EGCG synthase gene from tea was cloned. The full-length cDNA of methylated EGCG synthase gene was1040bp and open reading frame was738bp. By bioinformatics analysis, the amino acid residues base of methylated EGCG synthase gene was245, theoretic isoelectric point was5.39, molecular weight was27553.6Da. It is a kind of hydrophilic protein and transmenbrane protein. It has realistic meaning for improving tea cultivars resources by cloning the full-length cDN A of methylated EGCG synthase gene.The function of methylated EGCG synthase gene was confirmed by enzymatic test in vitro and Tua Prokaryotic Expression. Methylated EGCG synthase protein recombinant plasmid was constructed from pET-28-CsCOMT in tea plant. A recombinant protein in the expressive vector BL21was induced by IPTG From recombinant protein enzymatic test in vitro, the result shows methylated EGCG was induced successfully.A relative quantification’s analytic technique was used to analyze CsCOMT gene relative expression level.