丹参SmMYB36转录因子正负调控丹参酮和丹酚酸积累的差异机制

Tanshinone and phenolic acids,two groups of main active compounds in Salvia miltiorrhiza, are sometimes contrarily accumulated. MYB transcription factors are important regulators of tanshinone and phenolic acids accumulation. We found that SmMYB36 was a positive regulator of tanshinone accumulation and a negative regulator of phenolic acids accumulation. However, the regulation mechanism is still unclear. Based on the above research, the main activities of this project are as follows: (1) To analyze the induced expression pattern of SmMYB36 gene, and to reveal the relationship between SmMYB36 and "tanshinone and salvianolic acid contrarily accumulated ". (2) Analyze transcriptional activation and identify target genes of SmMYB39 by ChIP,EMSA and Y1H;(3) Identify the interaction and the binding domain of SmMYB39and SmbHLH by Yeast two hybrid, Co-IP and BiFC technology; (4) Revealing regulation mechanism of SmMYB39 and SmbHLH on tanshinone and phenolic acids biosynthesis by gene overexpression and RNAi.Finally, we will demonstrated differential mechanism of positive and negative regulation of tanshinones and phenolic acids in S. miltiorrhiza by SmMYB36 transcriptional factor. Our work will lay the foundation for uncovering the quality formation mechanism of S. miltiorrhiza and provid the theoretical basis for molecular breeding of S. miltiorrhiza.

丹参酮和丹酚酸是丹参的两类主要有效成分，二者具有显著的非同步积累现象。MYB转录因子是丹参酮和丹酚酸积累的重要调控因子，我们前期发现，SmMYB36转录因子对丹参酮积累具有极强的正调控作用，却显著抑制了丹酚酸积累，然而其分子机制尚不清楚。基于以上研究，本项目拟开展：①分析SmMYB36基因时空诱导表达模式，揭示SmMYB36与“丹参酮和丹酚酸非同步积累”的关系；②利用染色质免疫共沉淀、凝胶阻滞和酵母单杂交技术，鉴定SmMYB36靶基因、结合位点及转录激活功能；③通过酵母双杂交、双分子荧光互补和蛋白截短实验，体内外鉴定能够与SmMYB36互作的SmbHLH转录因子，确定互作区域；④通过过表达和RNA干扰技术，研究SmMYB36和SmbHLH互作调控两类成分积累的分子机制。最终阐明SmMYB36正负调控丹参酮和丹酚酸积累的差异机制，为揭示丹参品质的形成机制奠定基础，为丹参分子育种提供理论依据。

丹参酮和丹酚酸是丹参的两类主要有效成分，二者具有显著的非同步积累现象。MYB转录因子是丹参酮和丹酚酸积累的重要调控因子，我们研究发现，SmMYB36转录因子可以显著促进丹参酮类成分的积累及丹参酮生物合成关键基因的表达，抑制丹酚酸类成分的积累及丹酚酸生物合成关键基因的表达，进一步研究发现MYB36转录因子可以和丹参酮及丹酚酸合成关键基因启动子结合，调控这些基因的表达。基于该基因的研究，我们同时发现了MYB98b和MYB9a可以促进丹参酮的积累，MYB9a则抑制了丹酚酸积累。SmbHLH3对三种酚酸（咖啡酸、迷迭香酸、丹酚酸B）和四种丹参酮（隐丹参酮、二氢丹参酮Ⅰ、丹参酮Ⅰ、丹参酮ⅡA）成分的代谢都是负调控作用；SmbHLH7、SmbHLH148和SmbHLH130对酚酸类和丹参酮类物质的积累都是促进的作用；SmbHLH10对酚酸类成分代谢无显著调控作用，但促进了丹参酮类成分的生物合成。