MYB转录因子调控紫锥菊有效成分生物合成途径的分子机理及其应用研究

Echinacea purpurea (L.) Moench originates from the Midwest of North American with increasing immunity, anti-microbial, and anti-viral activities. It is one of the most important herbal of European and American areas. In the recent years, it was also introduced into China and cultivated in a large scale. Echinacea purpurea has obtained lots of attentions due to its great medicinal and economic value. Chicoric acid is the most important active ingredient of Echinacea purpurea, which has showed various of bioactivities. More and more evidences have exhibited it’s potential to be a drug. However, the biosynthesis pathway and regulation network of chicoric acid in plant is not clear. In our previous studies, we have identified the biosynthesis pathway of chicoric acid in Echinacea purpurea, which was generated from caftaric acid and chlorogenic acid. Meantime, it has been assumed that the lack of substrates limits the production of chicoric acid. Using co-expression analysis, we have obtained two new MYB transcription factors (EpMYB1 and EpMYB2) which were co-expressed with caftaric acid synthase (HTT) and chlorogenic acid synthase (HQT), respectively. The functions of these two transcription factors have been identified preliminary. In this project, we aim to identify the in vivo activities of EpMYB1 and EpMYB2 in Echinacea purpurea hairy root system. Then, we want to declare the regulation network of these transcription factors using transcriptome sequencing and other necessary methods. Last, the high chicoric acid production hairy root system need to be established using the identified transcription factors and inducible promoters. The whole project will lay the theory and practice foundations for the development of chicoric acid and Echinacea purpurea.

紫锥菊原产北美洲中西部地区，具有增强免疫力、抗菌、抗病毒等功效，是欧美地区最重要的草药之一。近年在我国也有大范围引种栽培，具有极高的药用和经济价值。菊苣酸是紫锥菊最重要的活性物质，表现出了多种生物活性，极具药物开发潜质，但是其生物合成途径和调控机制并不明确。申请人在前期工作基础中，鉴定了紫锥菊中菊苣酸的生物合成途径，并根据已有结果推测菊苣酸的合成受到了底物咖啡酰酒石酸和绿原酸水平的影响。利用共表达分析，我们筛选到了两个未报道的分别与咖啡酰酒石酸合成酶及绿原酸合成酶共表达的MYB转录因子EpMYB1和EpMYB2，并初步确认了功能。本课题拟在紫锥菊毛状根系统中进一步验证两个转录因子的功能，并结合转录组测序及其它多种手段解析转录因子作用机理和调控网络，同时利用可诱导型启动子，构建高产菊苣酸的毛状根系统，为菊苣酸次生代谢工程奠定基础。

紫锥菊是世界范围内广泛使用的药用植物之一，具有增强免疫力，预防和治疗感冒的作用。紫锥菊指来源于松果菊属的药用植物，我们利用活性追踪的方式成功解析了菊苣酸的生物合成途径，两个BAHD家族成员HTT和HQT分别催化咖啡酰酒石酸和绿原酸的合成，而两个产物被一个特殊的SCPL家族酶CAS催化合成菊苣酸。本研究系统比较了六个松果菊属植物的成分及活性差异，发现结构基因表达水平决定了菊苣酸的积累水平，预示着可以通过调控基因表达提高菊苣酸的含量。我们构建了紫松果菊不同组织部位的转录组及代谢组学数据，采用基于结构基因的共表达分析来筛选调控基因表达的转录因子。同时结合茉莉酸甲酯处理，紫外线处理测得的转录组数据，综合筛选到了若干个不同家族的转录因子。我们对其中的一个共表达且相应茉莉酸甲酯和紫外线处理的MYB家族转录因子EpMYB1进行了进一步研究，进化树分析发现其属于S2亚家族，定位于细胞核。通过双荧光素酶及酵母单杂交结合点突变，发现EpMYB1能够激活HQT基因的表达，并利用转基因方式确认了EpMYB1调控绿原酸的作用。总体上，我们围绕前期解析的菊苣酸生物合成途径，对其转录调控进行了系统研究，构建了紫松果菊的多组学数据，为后续进一步调控研究奠定了基础，并探究了紫松果菊转基因的可能性，为后期高菊苣酸品种的开发提供了基础。同时基于菊苣酸特殊的生物合成途径，对菊苣酸的生物学功能及变化规律进行了研究，更好的认识了菊苣酸的积累规律，为后期继续研究提供了参考。