茶树中ERF类转录因子对儿茶素合成调控的机理研究

The tea plant, Camellia sinensis (L.) O. Kuntze, is a perennial cross-pollination woody crop. Tea is made from tea plant leaves and is consumed as a popular natural non-alcoholic beverage worldwide due to its bioactive ingredients, including tea polyphenols, catechins, theanine, and polysaccharides. Catechins not only contribute to tea quality but also serve important functions in the anti-stress regulation of secondary metabolic pathways. Catechins are biosynthesized and accumulated in tea plant leaves, and their monomer contents also have high variability in different tea plant cultivars. Catechins biosynthesis in tea plant growth and development is important for the quality of tea. In higher plant, ERF family transcription factors are known to regulate diverse processes of plant growth and development, including the phenylpropanoid biosynthesis. The amount and composition of catechins are regulated by various kinds of transcription factors, including ERF family transcription factors. Till now, little information is available on the regulation mechanism of ERF family transcription factors involved in catechins biosynthesis in C. sinensis..On the basis of previous results, the project mainly aim to elucidate the regulation mechanism of catechins biosynthesis in C. sinensis through the results of catechins contents, expression profiles of involved genes, yeast one-hybrid system, transcriptomics, proteomics, transgenic plant. This study shall elucidated the regulation mechanisms and critical genes that involved in catechins biosynthesis in C. sinensis. The candidate genes include function genes in the catechins biosynthetic pathway and ERF family transcription factor genes. The main research areas were highlighted below: (1) the differences of catechins biosynthesis in different cultivars of C. sinensis; (2) expression profiles of the genes involved in catechins biosynthesis in C. sinensis; (3) expression profiles of the candidate ERF family transcription factor genes in C. sinensis; (4) the binding activity of candidate ERF family transcription factors in the up-stream sequence of catechins biosynthesis genes; (5) the function of those catechins biosynthesis genes and the candidate ERF family transcription factor genes be studied by transgenic Arabidopsis and transgenic tobacco plants. The researches in the project have a good foundation, and the contents and methods are innovative. The results will further deepen the regulation mechanism of ERF family transcription factors involved in catechins biosynthesis in C. sinensis. The project shall also provides a scientific basis for the improvement of quality of tea.

茶树是多年生木本植物，有很高的经济价值。儿茶素是茶叶的重要成分，具有多种保健功能。儿茶素主要在茶树叶片中合成和积累，不同茶树中儿茶素含量相差很大，儿茶素与茶叶品质密切相关。ERF类转录因子广泛参与植物生长发育的各个过程，也参与苯丙氨酸途径的代谢调控。目前ERF类转录因子调控茶树中儿茶素合成的机制还不清楚。项目在前期研究基础上开展ERF类转录因子对茶树儿茶素生物合成调控的机理研究。通过儿茶素测定、基因表达、酵母单杂交、转录组和蛋白质组学、植物转基因来研究茶树ERF类转录因子与儿茶素合成的关系。着重研究:茶树中儿茶素生物合成的时空差异;儿茶素合成相关基因的表达差异;茶树中ERF类转录因子基因表达差异; ERF类转录因子与儿茶素合成基因上游序列结合能力;转基因拟南芥和烟草验证茶树儿茶素合成和ERF调控关键基因的功能。项目有基础，有新意，深入研究茶树儿茶素合成调控机制，为茶树品质改良提供科学依据。

项目主要开展AP2/ERF家族转录因子对茶树儿茶素生物合成及类黄酮代谢途径调控的机理研究。通过儿茶素测定、基因表达、酵母单杂交、转录组和蛋白质组学、植物转基因等研究茶树AP2/ERF家族转录因子与儿茶素合成及类黄酮代谢途径的关系。检测分析了茶树中儿茶素生物合成的时空差异；儿茶素合成相关基因的表达差异；茶树中ERF类转录因子基因表达差异；酵母单杂交和转基因拟南芥和烟草验证茶树相关基因的功能。利用反相高效液相色谱对茶树中C、GC、EC和EGC的含量进行了检测。通过比较儿茶素组分比例和基因表达谱，发现三种关键酶基因（ANR、ANS和LAR）的表达可能影响了儿茶素在不同茶树品种间含量的差异。利用基因全合成的方法合成了上述三个基因，构建了三价植物表达载体，开展了拟南芥和烟草转基因研究。以茶树转录组和基因组数据为基础，鉴定出89个具有完整AP2结构域的茶树AP2/ERF转录因子，将其分为AP2、DREB、ERF、RAV和Soloist五个亚族。茶树中的CsAP2/ERF基因表达水平稳定。克隆了多个AP2/ERF家族转录因子基因，开展了CsERF12转基因拟南芥功能研究。分析了4个bHLH转录因子与儿茶素代谢的关系，对转录因子表达水平和儿茶素含量进行相关分析。利用iTRAQ技术，从茶树鉴定得到863个差异表达蛋白（DEPs），生物学功能以及蛋白-蛋白互作网络在不同的样本中呈现动态的变化过程。DEPs在萎凋过程中可能通过初级和次级代谢的调控影响了茶叶的颜色、滋味和香气。开展了相关差异蛋白与儿茶素代谢的相关性分析。开展茶树类黄酮代谢途径结构基因和调控基因对类黄酮代谢途径中的儿茶素和花青素合成调控研究。克隆茶树F3’H1基因和CsMYB2，茶树 ‘龙井43’和‘紫鹃’结构基因表达量和儿茶素、花青素含量进行关联分析，通过转基因研究推测在茶树类黄酮代谢途径起着重要的作用。项目有利于阐明茶树儿茶素合成和调控的机理，进一步解析转录因子对茶树儿茶素合成调控的分子机制，还有利于推动茶叶品质育种进程。项目取得的研究结果，为进一步开展茶树次生代谢相关分子生物学研究和育种工作提供了较好的基础。