Gene Expression Of Catechins Biosynthesis In Tea Plant [Camellia Sinensis(L.) O. Kuntze] With Real Time Fluorescence Quantitative PCR

Catechins biosynthesis mainly involves enzymes PAL, C4H, 4CL, CHS, CHI, F3H, F3'H, F3'5'H, DFR, LAR, ANS and ANR. Of them, LAR and ANR are directly interrelated with the synthesis of non-ether catechins C, GC, EC and EGC. Figuring out key genes influencing catechins biosynthesis is of great significance for the genetic engineering of tea secondary metabolism.In this study, the expressions of four common internal reference genes in different organs and tissues of tea plant were analyzed to find out those suitable for qRT-PCR. With fresh tea leaves as materials, expression differences of relevant structural genes and regulatory genes of catechins biosynthesis were studied by applying qRT-PCR. Changes of catechins concentrations, enzyme activity changes of DFR/LAR, ANR were figured out by using HPLC and enzyme detection, and the correlation between them was also discussed. Main findings were concluded as below.1. The expressions of four common internal reference genes (18SrRNA, GAPDH,β-Actin andα-Tubulin) in different organs and tissues of tea plant were investigated. The analysis with GeNorm and NormFinder algorithms revealed that in qRT-PCR analysis,β-Actin could be employed as a reference gene to study the gene expression in different organs and tissues and GAPDH as a reference gene to study the gene expression in different mature leaves and callus.2. The related genes'expression differences in catechins biosynthesis in qRT-PCR experiments were studied in leaves at different development stages. The results showed that the expression of PAL, F3H, LAR and DFR gradually decreased with the increase of leaf maturity. The expression of F3'H and C4H in the first leaf was the highest and the expression of F3'H and C4H gradually decreased with the increase of leaf maturity. The relevant expression levels of CHI and F3'5'H showed no obvious change from the first to the fourth leaf. The expression of ANR and ANS in buds and the third leaf was relatively higher. The expression of CHS and CsMYB2 from the first to the third leaf was highly expressed than in buds and old leaves. The expression of UFGT and CCoAOMT in the fourth leaf was the strongest.3. HPLC analysis showed that in fresh tea leaves at different growth stages, the accumulation of total catechins in the first leaf was the highest, bud was next, and the content of catechins in full mature leaf was remarkably less than that in the first leaf. The total content of catechins showed a similar changing trend with the expression of C4H and F3'H. The content of C, GC and EC were detected at low level and changed slightly in leaves at the different growth stages. By contrast, EGC content was raised gradually with development of leaves, while the content of EGCG and ECG were reduced gradually.4. Enzyme activity test suggested that C and GC, EC and EGC were direct products of DFR/LAR, and ANR respectively in tea plant, and the DFR/LAR activity gradually decreased with the increase of leaf maturity. In fresh tea leaves, the activity of DFR/LARDHM was higher than that of DFR/LARDHQ, the activity of ANR in the third leaf was the highest, and the activity of ANRCYA was far higher than that of ANRDEL. The changing laws of enzyme activity complied with the expression laws of relevant genes.