Cloning Of Polyphenol Oxidase CDNA Of Camellia Sinensis And Study On Transformation System Of Tea Plant

Tea plant (Camellia sinensis (L.) O. Kuntze), one of most importanteconomic plaflt in China, contains abundant secondary metabolic products, ofWhich polyphenols possess immense potential for food and rnedicalindustries due to their various physiological functions for human body. Thepolyphenol oxidases, as are well known, play vital roles in tea processing, e.g.high enzymatic activity is in favor of black tea while low enzymatic activitybenefits green tea. In present study, we fOcus on polyphenol oxidase (PPO)gene cloning and transfOrmation system of tea p1ant. The results werereported as follows.For the first time the PPO gene in tea plant was cloned. During Which,the consensus sequences of previously published PPO was used to design thedegenerate primers and the reverse tra-nscription and nest-PCR wereemployed. The obtained PPO gene of tea p1ant was registered in GenBank,Whose access number is AF269l92. Alignment of sequences with ClustalWindicates that the gene of tea has high homology with those found in otherplants, especialIy copper binding regions. The polyphenol oxidase of tea canbe clustered with most other woody plants according to phylogenetic treeanalysis.The effects of NAA and BAP at various concentrations supplemented toMS medium on tea stem culture were investigated with the number of newlydeveloped leaves as groWth index. It showed that the concentrations of NAAand BAP had great influences on the groWth of cultures. NAA at differentconcentrations showed no considerably different effects on the groWth ofcultures at early culture stage, until the stems were cultured for 50 days. Inthe contrast, BAP at various concefltrations exerted significant differenteffects on the groWth of cultures from the beginning of culturing. Furthermore, it was firstly proved that there t'as no interaction betWen BAP andNAA on the growth of culfored stem based on biostatistic ana1ysis. Thepresent study indicated the optimistic concendation for tea stem culture wasBAP 2.5-5 mg/L and NAA 0.5-l mg/L, under 1Vhich one stem at its heighcould develop about l5 leaves after culturing 60 days.The factors affecting the AgrObacterium tumefaciens-mediatedtransformation of tea leaf discs were studied based on GUS transieniexpression. It was found that callus fOrmation started to be depressed fromtea leaf discs cultured on MS rnedium containing 30 mg/L kana-mycin, thus50 mg/L kanamycin was considered suitable for selecting expression vectorwth neomycin phosphotransferase gene (NPT II ) Three Agrobacteriumtumefactens stains with different chromosomal background, LBA4404,EHA105, pGV3850, were tested. The results showed no considerablediffereflt in the infection between there thIee stains. During the process ofinfection, vacuum, on the contrary, weakened the transieflt eXPression ofGUS gene. In ligh of the resu1ts obtained in the present stUdy, the suitablesystem of Agrobacterium tumefaciens-mediated transfOrmation wassuggested that tea 1eaf discs be infected l0 min by LBA4404 of OD6oo about0.5-0.8 followed by co-culture of 2-3 days about 28 C in dadriess. Besides,AgNOs was found inhibiting the groWth of Agrobacterium tumefaciens, thuswas not suitable to be added into the co~culture rnedium.