| Caffeine (1,3,7-trimethylxanthine) is one of the most important components in tea plant. Moderate intake of caffeine is good for human health. However, excessive caffeine will have certain side effects to human body. In the domestic and international market, tea and tea products with low-caffeine and gradually popular. Conventional breeding methods of breeding low-caffeine tea varieties are too time consuming. The traditional chemical methods of removing caffeine in the process of machining cost too much, and may cause harmful residues in the tea, which has adverse effects on tea quality. As a result, These methods can not reduce caffeine content fundamentally to meet the market demand for low caffeine tea or tea drinks.The method of using genetic engineering can foster low-caffeine tea tree in a short time, and will not affect other biochemical composition in transgenic plants. Therefore, we have to do research and exploration of relevant genes from tea caffeine synthesis and metabolic pathways in more detail, so that we can use genetic engineering methods to modify and optimize relevant genes.The main caffeine biosynthetic pathway is a sequence consisting of xanthosine(XR)→7-methylxanthosine(7mXR)→7-methylxanthine(7-MX)→theobromine( Tb)→caffeine(Cf). Among them, There is no detailed report about the 7-methylxanthosine synthase in tea plant that catalyze the first step reaction. In addition, the research of tea caffeine synthase(TCS1) on protein levels is still limited and there is no sale for the commercialization of TCS1 antibody.In this study, we tried to find the 7-N-methyltransferase(7-NMT) to control the output of 7-MX, so as to regulate the biosynthesis of caffeine. Meanwhile, we prepared the TCS1 polyclonal antibody for studying tea caffeine synthesis(TCS1) on protein levels. The main research contents and results are as follows:1. According to the cDNA full-length sequence of CaXMT1, we found out the complete ORF (Open Reading Frame) and designed the specific primers. The fragment was amplified by RT-PCR, which was subsequently cloned into pMD-18T vector for sequencing analysis. The results of sequencing analysis showed that the nucleotide sequences had high identity (up to 99%) with the CaXMT1-ORF sequence.2. The primers for prokaryotic expression were designed and synthesized. The prokaryotic expression plasmid pGEX-4t-NMT was constructed successfully. The recombinant protein was expressed by induction with IPTG. Then we tested in vitro enzyme activity. The results show that induced by optimizing the conditions, we obtained the best expression condition of the recombinant protein as:28℃,6 h. Soluble protein expression is also expressed in but the quantity is not high. The testing results of HPLC and LC-MS were, suggesting the recombinant proteins can directly catalyze the conversion from XR to 7-MX.3. Tea caffeine synthase (TCS1) was cloned from the leaf of Camellia sinensis. The recombinant protein was proved to catalyze the last two steps of caffeine synthesis and subsequently purified by affinity chromatography. The TCS1 antibody was further refined by immunizing white rabbits with the purified protein. ELISA detection of the antibody recommended dilution ratio of 1:2000. We verified the favourable specific of the antibodies by western blot. |
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