EST analysis of genes involved in secondary metabolism in Camellia sinensis (tea), using suppression subtractive hybridization

Abstract

Camellia sinensis (tea) is a commercially important crop that is valued for its secondary metabolites. Higher levels of catechins are accumulated in young leaves than in mature leaves. To understand the molecular regulation of secondary metabolism in tea leaves, differentially expressed genes of interest should be identified, cloned, and studied in detail. A total of 588 cDNA clones from a subtractive cDNA library were randomly picked, sequenced, and analyzed. High-quality sequences were isolated for 508 of these clones. BlastX comparisons indicated that about 8.7% of the clones encoded enzymes involved in secondary metabolism, with a particularly high abundance of flavonoid-metabolism proteins (5.1%). These ESTs facilitated the isolation and characterization of genes involved in the flavonoid pathway in tea: chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3′5′-hydroxylase (F3′5′H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin reductase (LCR). The three genes encoding F3H, DFR, and LCR were more highly expressed in young leaves than in mature leaves. These results indicate that flavonoid biosynthesis genes from tea are differentially regulated in the developmental stages. The subtractive cDNA library and EST database described in this study represent a valuable resource for future research aimed at improving economically important secondary metabolic characteristics in crops.