SmPAP1-SmMYC复合体调控丹参酚酸类活性成分合成的机理研究

The phenolic acids are main active ingredients of S.miltiorrhiza. From our previous results, the expression of transcription factors SmPAP1 and SmMYC is correlated well with the accumulation of phenolic acids in S.miltiorrhiza, and SmPAP1 potentially regulates SmMYC expression. Accordingly, it is speculated that there may exist a regulation mode that SmPAP1-SmMYC complex regulate expression of the enzyme genes targeted and subsequently control phenolic acids biosynthesis, and activate SmMYC per si expression. However, the detailed interaction between the SmPAP1 and SmMYC and the mechanism for their subsequent coordinate regulation on phenolic acids biosynthesis still remain to be determined. In this proposal, we are planning to validate SmMYC as a key transcription factor governing phenolic acids biosynthesis of S.miltiorrhiza by SmMYC-specific silencing, and define the interaction between SmPAP1 and SmMYC using yeast two-hybrid combined with BiFC assay. Subequently, electrophoretic mobility shift assay (EMSA) and DNAase I footprinting will be employed to unravel the binding of SmPAP1 to specific region within SmMYC promoter. Besides, transcriptional activation of SmMYC itself and enzyme genes involved in phenolic acids biosynthesis under the control of SmPAP1-SmMYC complex will be investigated using transient promoter activation assay. Furthermore, expression level of the enzyme genes involved in phenolic acids biosynthesis and contents of phenolic acids will be assessed in the 35S:SmMYC or 35S:SmPAP1, as well as co-expression of SmMYC and SmPAP1 transgenic lines compared with control. Our studies aim to uncover the molecular mechanism for regulation of the phenolic acid biosynthesis in S.miltiorrhiza by SmPAP1-SmMYC complex. The success of proposal will provide strategy for practicable genetic modification of S.miltiorrhiza.

酚酸类化合物是丹参重要的活性成分。我们发现转录因子SmPAP1及SmMYC与酚酸类成分的合成密切相关，SmPAP1对SmMYC的表达具有潜在的调控作用。推测丹参中存在SmPAP1-SmMYC复合体调控酶基因的表达进而控制酚酸类物质的合成，同时也调控SmMYC自身的表达，但其分子机制尚不清楚。本课题拟通过沉默SmMYC验证其在丹参酚酸类化合物合成中的关键作用；利用酵母双杂交和双分子荧光补偿法明确SmPAP1与SmMYC的相互作用；采用DNA酶I足迹、凝胶迁移率技术以及启动子瞬时激活体系研究SmPAP1对SmMYC启动子的特异结合及转录激活方式，并明确SmPAP1-SmMYC复合体作用于丹参酚酸类化合物合成途径的酶基因；进一步在丹参中进行SmPAP1及SmMYC的过表达或共表达。研究结果有望阐明SmPAP1-SmMYC调控丹参酚酸类活性成分合成的分子机制，为丹参高效、可行的改良提供思路及策略。

丹参酚酸类化合物是丹参中的主要活性成分，由苯丙烷类和酪氨酸代谢途径产生。目前对丹参酚酸类化合物合成途径的调控机制研究较少。我们前期研究结果显示丹参酚酸类化合物合成途径上的酶基因与SmPAP1及SmMYC在表达水平上均呈现显著性正相关，但是SmPAP1与SmMYC如何调控这些酶基因的表达及其具体作用方式仍不清楚。基于以上目标，本课题主要开展了以下研究工作：1.通过对SmMYC进行了特异性基因沉默，分析了SmMYC基因沉默株相应组织中酚酸类化合物代谢途径上酶基因表达水平以及酚酸类化合物含量的变化，验证SmMYC与丹参酚酸类化合物合成的相关性；2.依次通过进行酵母双杂交以及烟草细胞内双分子荧光补偿法检测实验，检测细胞内SmMYC与SmPAP之间是否存在蛋白相互作用；3.为了进一步探究丹参体内SmPAP1对SmMYC的转录调控作用，课题组首先利用凝胶电泳迁移率检测（EMSA），分析了体外SmPAP1与SmMYC启动子特异位点的结合情况，同时进一步采用启动子瞬时激活检测分析了SmPAP1过表达后SmMYC基因启动子的转录激活情况；4.还我们检测了调控蛋白与酶基因启动子序列的位点特异性结合性，以确认SmPAP1-SmMYC转录复合体对酶基因的表达调控模式。实验结果显示，丹参体内SmMYC的确作为一个重要的转录因子参与了丹参酚酸类次生代谢产物合成的调控；在丹参体内确实存在着SmMYC与SmPAP1的蛋白相互作用，二者通过形成了一个转录复合体存在；凝胶电泳迁移率检测（in vitro）以及启动子瞬时激活检测(in vivo)研究结果显示，SmPAP1不但对SmMYC的启动子具有激活作用，而且与SmMYC启动子的相应顺式作用DNA元件能进行特异性结合，说明在丹参体内SmPAP1的确作为SmMYC启动子特定区域进行特异性结合。此外，我们还研究了丹参JAZ蛋白对酚酸类化合物合成的调控作用，并利用组合基因改造的方法获得了高酚酸含量的丹参品系。同时课题组通过遗传操作获得了高抗性及高丹参酮含量丹参。为丹参次生代谢物质合成调控提供新的策略，且为丹参分子育种提供研究基础。