Tea Tree Terpenoids Metabolism Upstream Metabolic Precursor Gene Expression Regulation System Of Genetically Modified (gm) And Tea Tree

Tea is universally popular with the consumer at home and abroad for its’health function and the smell of pleasant. Volatile terpenoid compounds are important components and dominate quality and flavor of Camellia sinesis. Terpenoids metabolic mechanism and further regulation of C. sinensis need turn to transgenic technology. At present, transgenic technology of tea tree is not mature and gaining the transgenic tea tree is hard. In this study, genome and transcriptome sequencing data were used to identify the gene components in the early stages of biosynthetic pathway for terpenoid production in C. sinensis. Furthermore, feeding experiments with precursor metabolites to callus and leaf disc cultures were conducted for identifying the genes whose expression is mediated by the precursor metabolite. Moreover,this experiment using agrobacterium transformed tea tree callus, and trying to build up a genetically modified system in tea tree.1. In this study, seven genes in the upstream terpeonoid pathway leading to the biosynthesis of isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), and farnesyl pyrophosphate were identified in Camellia sinensis through BLASTX search using previously obtained sequencing data. The identified genes encode cetyl-CoA C-acetyl transferase (AACT),3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), and3-hydroxy-3-methylglutaryl-CoA reductase (HMGR),1-deoxy-D-xylulose5-phosphate (DXP) reductoisomerase (DXR),4-(cytidine5’-diphospho)-2-C-methyl-D-erythritol kinase (CMK),1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), isopentenyl diphosphate isomerase (IDI), and farnesyl pyrophosphate synthase (FPPS). Metabolite-mediated gene expression analysis was performed through precursor feeding experiments followed by real-time PCR analysis. Results showed that acetyl-CoA, the starting metabolite of the MVA pathway, induced1.5-,3-, and2.5-fold increases in the transcript level of CsAACT, CsHMGS, and CsHMGR, respectively; and DXP, the first intermediate of the MEP pathway, induced a1.7-,2.3-and1.5-fold increases for CsDXR, CsCMK and CsHDS. The CsIDI, and CsFPPS genes all had an approximately2-fold increase when fed with DXP, IPP and DMAPP, but not with acetyl-CoA. This study has identified the metabolite-mediated expression patterns of several genes at the early stages of terpenoid biosynthetic pathway in C. sinensis.2. Calli were induced from leaves of tea cultivar’Nongkangzao’, and those growing vigorously with compact tissue were chosen for agrobacterium-mediated transformation by EHA105. The transgenic callus was screened using hygromycin. The resistant callus was confirmed by RT-PCR and GUS dyeing. The results showed that transgenic callus was obtained using this method, and the conversion rate was about10%after adding100μmol/L acetosyringone (AS). The results also showed that the efficiency of agrobacterium-mediated transformation can be effectively improved by adding AS, but the increase in osmotic pressure had no effect.The results has the important theoretical significance and application value to reveal volatile sesquiterpanes alcohols material synthesis and gene regulation technology of C. sinensis and improve the fragrance of a tea quality.