Genes Cloning And Function Identification Of Polyphenol Oxidases In Camellia Sinensis

Polyphenol oxidases(PPOs)are copper-binding enzymes,which play a crucial role in the processing of tea.Their synthesis mechanism in tea plants is that they can oxidase catechins into theaflavins and thearubigins.These products are key quality components especially have an important influence in black tea.According to the literature,polyphenol oxidases are mainly related to the stress resistance of plants.However,they are not clear whether polyphenol oxidases are involved in resistance of external stress or the secondary metabolic pathways of plants during the growth of tea plants.Therefore,it has great theoretical and practical significance to explore the physiological functions of polyphenol oxidases in tea plants.Three genes were successfully cloned in the transcriptome data analysis of tea plant.Bioinformatics analysis of three genes were performed by DNAMAN and MEGA5.0 software,the results show that the functions of three genes were more likely associated with the resistance of the abiotic stress in tea plant.We analysed the vivo subcellular localization and they were all located in the chloroplast.The relative expression level of CsPPOs in different tea organs,as well as the various inductive treatment by using qRT-PCR analysis.Three genes were all expressed in different organs of tea plant.The expression level of CsPPO1 was significantly high in the root.And the expression level of CsPPO3 in mature organs are higher than in the young organs.We also found that CsPPO1 and CsPPO3 were more likely related in the anti-virus defense mechanism in tea plant.Moreover,CsPPO1 and CsPPO2 had closer relationship with the response to light induction of tea plant.Through the construction of prokaryotic expression vector of CsPPOs,they were transformed into the E.coli Novablue(DE3)and successfully induced CsPPOs protein.Three recombinant CsPPOs proteins could participate in catalyzing catechins and we could detect the products at 380 nm respectively.Finally,the overexpression of transgenic tabaccos were used to detected the secondary metabolites of the tea polyphenol oxidases.On the phenotype,the flowers of transgenic tabaccos were more reddish than nontransgenic tabaccos(G28).And other metabolic substances in transgenic tabaccos also changed.This paper aims to explore the physiological significance of tea polyphenol oxidases through the above studies.It provides new ideas for tea plant breeding and the improvement of tea quality.