Gene Expression Of Methylated EGCG Biosynthesis With Real Time Fluorescence Quantitative PCR&Study On Analysis Method Of Thin-layer Chromatography

Methylated catechin is important secondary metabolites of tea plant, which possesses strong anti-allergic activities. However, the mechanism of methylated catechin biosynthesis in tea plants is largely unknown due to the scarce tea resources with high content of methylated catechin. In this paper, the efficient thin-layer chromatographic separation method of methylated EGCG and catechins is investigated. Using the tender tea leaves of camellia sinesis Jinmudan and Jinguanyin with high methylated EGCG as explants, the tea callus was induced and suspension. The differences of methylated EGCG were analyzed by HPLC in different tea cultivar leaves, tea leaves with different season and tea leaves with different maturity. The related genes’ expression differences in catechins biosynthesis in qRT-PCR experiments were studied in different tea cultivar leaves, tea leaves with different season and tea leaves with different maturity. By investigating the relationship between gene and methylated EGCG, the aim of this study is to provide theoretical support for regulation of methylated EGCG biosynthesis.. The main results of this study are presented as follows:1. In this study, with polyamide serving as adsorbent, the author conducts thin-layer chromatography for two methylation EGCG and5kinds of catechins. The use of methanol-acetic acid (2:3, v) solvent system enables the favorable separation of EGCG3Me, EGCG4"Me", EC, C, EGC, ECG and EGCG. The applicability of the developed method was checked for separation of the extracts of tea with high methylated EGCG content.2. The related genes’ expression differences in catechins biosynthesis in qRT-PCR experiments were studied in different tea cultivar leaves, tea leaves with different season and tea leaves with different maturity. The results showed that the expression of CHS in spring Jinguanyin cultivar leaves was the highest&the expression of PAL, C4H,4CL, CHI, DFR, F3H, F3’H,F3’5’H, LAR, ANS, ANR and CsCOMT in autumn Jinguanyin cultivar leaves was the highest. The expression of PAL, C4H,4CL, DFR, CHS, CHI, F3H and F3’5’H in spring Jinmudan cultivar leaves was the highest. The expression of F3’H, ANS and ANR in summer Jinmudan cultivar leaves was the highest&LAR and CsCOMT in autumn Jinmudan cultivar leaves was the highest. The expression of C4H,4CL, CHS, LAR and ANR gradually increased with the increase of leaf maturity. The expression of CHI from the first to the third leaf was highly expressed than in buds and the fourth leave. The expression of PAL in first and the fourth leaf was relatively higher and the expression of ANS in the fourth leaf was the highest. The expression of CsCOMT, DFR and F3’5’H’in the second leaf was the highest. The expression of F3’5’H in the first and the third leaf was relatively higher&the expression of F3H in the second and the fourth leaf was relatively higher.3. HPLC analysis showed that EGCG3’Me content was highest in autumn Jinguanyin cultivar leaves&EGCG3’Me content was highest in summer Jinmudan cultivar leaves. EGCG3’Me content was increased gradually with development of the first two leaves and the accumulation of EGCG3’Me in the second leaf was the highest. Then the content of EGCG3’Me were reduced gradually&EGCG3’Me in the fourth leaf was the lowest.4. Using the tender tea leaves of camellia sinesis Jinguanyin as explants, the induced rate of MS(A) medium is relatively higher. Use the second tea leaves as explants,35%sodium hypochlorite, soak for18minutes, could be obtained better efficiency of the induction. In the subculture, the catechin content of Camellia sinensis var. Jinmudan callus was the highest among tea callus. Except Camellia sinensis var. Huangmeigui, the catechin content in the other five tea varieties using B5culture medium was higher than that using the MS culture medium. When the callus cultured from Camellia sinensis var. Jin mudan callus were used in the B5culture medium and at2,4-D0.5mg/L and KT1.0mg/L, we obtained the maximum catechins production.This paper attempts to get suspended training seeds by using cellulose enzyme and pectin enzyme for digestion. After several subcultures, suspended cell lines is acquired by taking A-2method.