DcMYB1和DcbHLH5协同调控海南龙血树类黄酮生物合成的分子机制

As a traditional Chinese medicine, dragon's blood has various pharmacological properties such as eliminating stasis to activate blood, anti-inflammatory analgesic activities and so on. Hainan Dragon trees (Dracaena cambodiana) are the main plant resources of dragon's blood in China, and the main chemical constituents of the dragon's blood from which are flavonoids. The recent studies about dragon's blood mainly focus on the chemical constituents and pharmacological activities, while the molecular mechanisms of dragon's blood formation remain unknown. In previous studies, the project team carried out the research on the mechanism of flavonoid accumulation and induced formation of dragon's blood in D. cambodiana. We found that the expression of DcMYB1 and DcbHLH5 was positively correlated with the accumulation of flavonoids, DcbHLH5 could bind to the promoters of flavonoid biosynthesis genes DcCHS1 and DcCHI1, and DcbHLH5 interacted with DcMYB1, which indicating that DcMYB1 and DcbHLH5 may cooperatively regulate the biosynthesis of flavonoids. The project will study molecular mechanisms underlying the coordinate regulation of flavonoid biosynthesis by DcMYB1 and DcbHLH5: focus on the mechanism of DcMYB1, DcbHLH5 and their interaction in regulating the key enzyme genes of flavonoid synthesis. The implementation of the project will provide a foundation for further research on the flavonoid accumulation and induced formation of dragon's blood, and also provide the theoretic foundation for increasing the yield of dragon's blood through biotechnology in D. cambodiana.

血竭是一种传统中药，具有活血化瘀、消炎镇痛等作用。海南龙血树是国产血竭的主要基源植物，其血竭主要化学成分为类黄酮化合物。目前对血竭的研究集中在化学成分及药理活性分析，对血竭形成的分子机制并不了解。项目组开展了海南龙血树类黄酮积累及血竭诱导产生机制的研究。发现转录因子DcMYB1和DcbHLH5的表达与类黄酮积累正相关，DcbHLH5能够与类黄酮合成关键酶基因DcCHS1和DcCHI1的启动子结合，且DcbHLH5与DcMYB1互作，表明DcMYB1和DcbHLH5可能协同调控类黄酮的生物合成。本项目将研究DcMYB1和DcbHLH5协同调控类黄酮生物合成的分子机制，重点探讨DcMYB1和DcbHLH5以及它们之间相互作用对类黄酮合成关键酶基因的调节机理，为揭示海南龙血树类黄酮积累和血竭诱导产生的机制奠定基础，也将为通过生物技术提高海南龙血树中血竭的产量提供理论依据。

血竭是一种传统中药，具有活血化瘀、消炎镇痛等作用。海南龙血树是国产血竭的主要基源植物，其血竭主要化学成分为类黄酮化合物。目前对血竭的研究集中在化学成分及药理活性分析，对血竭形成的分子机制并不了解。本研究对血竭形成前后茎干中化学成分变化的分子基础进行了研究；对类黄酮生物合成相关基因进行了挖掘；对DcbHLH5和DcMYB1在类黄酮积累中的功能进行了鉴定；对DcbHLH5、DcMYB1和DcWD40-1协同调控类黄酮积累的机制进行了探索。研究发现在自然生长情况下海南龙血树干中的化合物主要是甾体皂苷，而在形成血竭后，主要是类黄酮化合物。转录组学分析结果显示在血竭形成过程中类黄酮生物合成相关酶基因的表达量显著增高，而甾体皂苷生物合成相关酶基因的表达量显著下降，与代谢产物的变化相一致，甾体皂苷和类黄酮在血竭形成过程中存在此消彼长的现象。初步鉴定10个结构基因受到血竭诱导剂的显著诱导，可能在海南龙血树类黄酮生物中具有重要的调控作用，并进一步通过表达调控分析、体外酶活分析和瞬时过表达等技术鉴定DcCHI1/4是类黄酮积累关键基因。通过进化和表达分析筛选到DcbHLH5、DcMYB1和DcWD40-1可能在海南龙血树类黄酮积累中具有重要的调控作用。DcbHLH5和DcMYB1定位于细胞核，在海南龙血树中瞬时过表达或在烟草中过表达均能促进类黄酮的积累。DcbHLH5能够能够与DcCHS1,DcCHS2和DcCHI1的启动子结合并增强其活性，DcMYB1能增强DcCHS2和DcCHI1的启动子活性。DcbHLH5与DcMYB1和DcWD40-1转录因子能够相互作用形成DcMYB1-DcbHLH5-DcWD40-1复合体协同调激活DcCHS1,DcCHS2和DcCHI1的表达参与对海南龙血树生物合成的调控。项目在执行过程中发表论文7篇，其中SCI期刊论文4篇；培养研究生2名。本研究结果为进一步揭示海南龙血树类黄酮积累和血竭诱导产生的机制奠定基础，也将为通过生物技术提高海南龙血树中血竭的产量提供理论依据。