箭叶淫羊藿叶片发育过程中花青素苷和黄酮醇苷代谢分支的分子调控

Because of its unique structural and functional activity, the main active ingredient icariin in traditional Chinese medicine herba epimedii (prepared from arial parts of Epimedium species) has been singled out as an important class of flavonol glycosides. On the one hand, Epimedium wild resources reduced dramatically due to the over harvesting in the past decade, on the other hand, the domestic and foreign demand of herba epimedii has been increasing considerably with the discovery of new functions (such as anticancer, the treatment of asthma and other new features). Therefore, in-depth study of icariin biosynthesis and molecular control mechanism is in urgent need to lay an important theoretical basis for the biosynthesis of flavonol glycosides in Epimedium species, as well as the molecular breeding and cultivation. Combining leaf transcriptome sequencing, suppression subtractive hybridization library, and homologous cloning method, the genes including structural genes and regulatory factors relating to the flavonoid biosynthetic pathway will be cloned, especially those for the anthocyanin and flavonol glycosides branches. The relationship between gene expression and accumulation of chemical composition will be explored during the leaf development from young red leaves to mature green leaves. The function of transcription factors will be characterized based on the gene expression, gene transformation, interaction between the TFs and promoters of important structural genes, and interaction between different transcription factors. The competition and molecular regulation mechanism between the anthocyanin and flavonol glycosides metabolic branches will be illustrated in E. sagittatum.

传统中药材淫羊藿中主要的活性成分淫羊藿苷等因其独特的结构和功能活性，已经被单独列为一类重要的黄酮醇苷。一方面由于过渡采收野生淫羊藿造成中药材资源急剧减少，另一方面随着抗癌、治哮喘等新功能的发掘国内外对淫羊藿的需求不断增加。因此，急需深入研究淫羊藿苷的合成和分子调控机制，为淫羊藿黄酮醇苷的生物合成、淫羊藿的分子育种、人工栽培奠定重要的理论基础。本项目通过箭叶淫羊藿叶片转录组测序、差减文库、同源克隆等方法克隆类黄酮代谢途径的主要结构基因和调控因子，重点解析下游花青素苷和黄酮醇苷的代谢分支；通过分析幼嫩红色叶片到成熟绿色叶片发育过程中基因表达和化学成分积累的关系，鉴定花青素苷和黄酮醇苷两个代谢分支的重要结构基因，及发掘调控这两个代谢分支的转录因子；研究转录因子的基因功能、与结构基因启动子的互作、不同转录因子之间的互作，解析淫羊藿中类黄酮代谢下游的花青素苷和黄酮醇苷代谢分支的关系和分子调控机制。

本项目的实施完全按照原计划内容进行。通过本项目的实施，我们基本完成了对箭叶淫羊藿类黄酮生物合成途径的解析，克隆得到了大部分途径相关的结构基因；并且分离得到了17个MYB转录因子和2个bHLH转录因子；并对其中的4个调控类黄酮合成途径的MYB转录因子进行了深入功能分析。另外，我们还推测出花青素和黄酮醇代谢分支途径之间可能存在协同作用关系，受到MYB-bHLH-WD40 复合体的协同调控。总体来说，项目执行情况超额完成了原定目标，共发表论文SCI10篇（7篇本基金排序第一，3篇本基金排序第二）、中文一篇（本基金排序第一）、书的综述章节一篇、培育了国家级淫羊藿优良品种3个、申请专利2项。该项基金的研究成果为淫羊藿的分子遗传和育种、主要活性成分的生物合成、优良栽培品种培育和规模化栽培奠定了坚实的理论基础。以此基金委基础的淫羊藿苷生物合成正在进行，对于满足生物制药公司的化合物需求具有重要意义；培育出的淫羊藿3个优良品种已经受到多家公司的关注，开始了规模化栽培。总之，该项目的实施及相关成果，是淫羊藿资源保护和可持续开发利用的一个重要里程碑。