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

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.

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。

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NMT表达的时空特异性表明其表达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，拟克隆yhNMT1基因启动子，通过文库构建与筛选获得合成酶基因yhNMT1的转录因子，并对获得的转录因子与靶基因启动子的结合进行验证，利用烟草瞬时表达体系表明TF对靶基因的调控作用，并借助茶叶愈伤组织转基因技术揭示TF对茶叶咖啡碱合成的调控作用机理。研究结果：克隆获得的yhNMT1启动子长2210bp，分析揭示其具有G-box等与特定转录因子识别的元件；通过文库构建筛选和转录组学比较分析共克隆获得yhTF1等10个转录因子，其中除yhTF4外的9个转录因子表达蛋白亚细胞定位于细胞核，并发现转录因子MYC2b与WRKY6、GBF2、CPRF1之间存在蛋白互作；在烟草瞬时表达体系中，yhTF1、yhTF3、yhTF4 、WRKY1、MYB306均具明显促进yhNMT1表达的功能，yhTF2无明显的调控作用， CPRF1、GBF2、MYC2b和WRKY6对yhNMT1表达具抑制作用， MYC2b与互作蛋白GBF2、CPRF1、WRKY6联合对yhNMT1表达具更强的抑制作用；茶叶愈伤组织转基因鉴定表明，不同转录因子对yhNMT1表达和咖啡碱合成具有不同的调控模式，其中yhTF1、CPRF1、GBF2、MYB306、MYC2b具有反向调控yhNMT1表达水平和咖啡碱合成作用，yhTF2、yhTF3、yhTF4、WRKY1具有正向调控yhNMT1表达水平和咖啡碱合成作用。本研究的开展及取得的结果，从转录因子角度深入揭示了茶叶咖啡碱合成的分子调控机理，更为茶叶的特定含量咖啡碱育种提供技术与理论参考。