基于ChIP的茶叶咖啡碱合成酶转录调控因子的分离及其对咖啡碱合成的调控机理

咖啡碱是茶树重要次生代谢物，体内合成主要由关键酶基因NMT催化底物黄嘌呤核苷经三步转甲基化反应生成。茶叶中大量NMT基因已被克隆和功能鉴定，但同叶位不同NMT具差异表达水平及不同叶位同基因表现时空特异性，表明咖啡碱合成不仅受合成酶基因NMT调控，还受相关调控因子尤其转录因子TF调控，开展TF对茶咖啡碱合成调控研究具重要意义。本研究以英红9号为材料，通过文库构建克隆NMT基因启动子，利用国际分析软件和TF数据库，克隆获得候选TF基因并通过ChIP等技术进行鉴定，利用免疫组织化学和酵母双杂交分析TF的亚细胞定位及TF间互作，通过TF基因超表达和RNAi技术对茶叶转基因，分析转基因前/后愈伤组织中TF和NMT表达、TF结合NMT启动子情况及咖啡碱含量等变化，获得TF调控咖啡碱合成的内在关系。项目的开展将从转录调控因子角度深入揭示茶叶体内咖啡碱生物合成调控的分子机理。

Transcription factors play a key role in modulate the expression of functional target gene and affect the biosynthesis of functional product. Caffeine (1,3,7-trimethylxanthine) is an important secondary metabolite in tea (Camellia sinensis) and different tea varieties showed different caffeine content. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine biosynthetic pathway as the major route to caffeine. N-methyltransferase, the SAM-dependent methyltransferase involved in the methylation of purine ring in the last three steps of caffeine biosynthesis, play an important roles, and a total 19 functional genes related to the production of caffeine isolated from tea plants in our previous work, and the expression of NMT present typical temporal and spatial specificity in tea plants, suggest the biosynthesis of caffeine modulated not only by NMT genes but also some other modulate factors , especially modulated by transcription factors involved in caffeine biosynthesis, which hint the importance to reveal the transcription factors and the role of transcription factors in control the biosynthesis of caffeine in tea plants. In this research project, the tea plants of Yinghong 9 was used, and the main work as follows: (1) to isolate the genes of transcription factors which target caffeine biosynthetase gene NMT base on the cloned promoter of NMT and analysised by bioinformatics; (2) to identify the transcription factors binding to the promoter DNA of NMT gene by the use of technique of ChIP in live tea cell and make sure how does the transcription factors binding to promoter modulate the transcription of downstream gene by tobacco transgene ; (3) to identify the exist place of transcription factors protein in tea cell and the quantity change of transcription factors protein for the protein transfer among the sub-cell by the use of Immunohistochemical staining method; (4) to analysis whether there exist gene interaction among different transcription factors by the technique of yeast two hybridization; (5) to understand how does the transcription factors modulate the biosynthesis of caffeine by affect the expression of target gene NMT, the transgene was conducted including gene overexpression and RNAi of transcription factors on callus induced from tea, and the expression level of both the transcription factors and NMT gene were analysised by qPCR and western blot in genetic modified callus, and the caffeine content was determined by HPLC in callus. This work will focus on the isolation the transcription factors gene of NMT and how does the transcription factors modulate the caffeine biosynthesis by affect the expression of NMT and help us to know the molecular mechanism of caffeine metabolite in tea plants.