Differential Gene Expression Of Endogenous Glycosidases And Caffeine Biosynthesis Enzymes Induced By Pathogenic Fungi Infection In Leaves Of Camellia Sinensis

Glycosides are the main form of the volatiles such as monoterpene alcohols and aromaticalcohols in tea leaves, and most of glycosides are primeverosides and glucosides. Enzymesresponsible for biosynthesis and hydrolysis of glycosides mainly include β-Primeverosidase,β-Xylosidase, β-GlucosidaseⅠ, β-GlucosidaseⅡ and β-Glucosyltransferase. The volatileaglycones released from glycosides hydrolysis are not only the material base of tea aroma, butalso participate in the tea plant defense reaction to disease and pests. The analysis ofdifferential gene expression of endogenous glycosidases induced by pathogenic fungiinfection in tea leaves can widen our horizon of interactions between tea plant and nature, andsupport the further study of tea resistance mechanism. It also provides the valuable theory thatwill be a great help to improvement of tea aromatic quality.As the main constituent of purine alkaloids in tea plant, caffeine attracts close attentionbecause of its pharmacological effects. However, the study of interactions between caffeineand physiological resistance is really rare. Now it is reported that caffeine, as an effectivefungistatic material, can inhibit the growth of common pathogens and play a role in tea plantdefense reaction to disease. The analysis of differential gene expression of caffeinebiosynthesis enzymes induced by pathogenic fungi infection in tea leaves can ascertain thecaffeine’s fungistatic mechanism and develop the resistance theory further.In this paper, differential gene expression of endogenous glycosidases and caffeinebiosynthesis enzymes induced by pathogenic fungi infection in tea leaves were studied, usingreal-time fluorescence quantitative PCR (qRT-PCR) technology with GAPDH used as areference gene. And changes of tea caffeine content at different stages of fungal infectionwere detected by high performance liquid chromatography. The main results are as follows.Results of real time quantitative PCR of endogenous glycosidases showed that the geneexpression levels of β-Primeverosidase, β-GlucosidaseⅠ, β-GlucosidaseⅡ andβ-glucosyltransferase had consistent trends in three cultivars and got up-regulation to varyingdegrees. Up-regulations of these key genes probably mean accumulation more glycosidicaroma precursors release a lot more volatile aglycones, and it will be helpful for strengthendisease resistance of tea plant. And up-regulations usually occurred in the early stage ofinfection indicated that mild infection might stimulate the accumulation of precursors andrelease of aglycones.Results of real time quantitative PCR of caffeine biosynthesis enzymes showed thatTCS1, sAMS, TIDH had the consistent trend in three varieties. The up-regulation of TCS1was significant and sAMS was also obvious, while TIDH had no significant change. TCS1is responsible for last two steps of methylation of caffeine biosynthesis, catalyzing from7-methylxanthine to theobromine, from theobromine to caffeine. sAMS can activatemethionine into adenosylmethionine, and the latter is the sole source of methyl.Up-regulations of these key genes probably mean biosynthesis and accumulation of morecaffeine.Results of HPLC of caffeine showed that the caffeine content of two varieties shuchazaoand duokangxiang had similar ups and downs. They appeared to be lower in the early stage,growth in middle stage and then drop again in the later stage. No consistent relations betweenthe differential expression and the content of caffeine was observed, because it is acomplicated process from gene expression to enzymatic activity, and then to caffeinebiosynthesis. The final content of caffeine in tea plant is affected by many factors in themetabolic process.