丹参SmMYB97通过JAZ8/DELLA1调控丹酚酸B的合成机制研究

In our previous studies, SmMYB97 was regulated by SmJAZ8 of JAs signaling pathway in Salvia miltiorrhiza, wihch enhanced the gene expression by combining SmPAL1 and SmTAT1 promoters to involve in the regulation of salvianolic acid B synthesis. On the other hand, SmMYB97 interacted with SmDELLA1 belong to the GAs signaling pathway, and SmDELLA1 had been confirmed to affect the biosynthesis of salvianolic acid in S.miltiorrhiza. SmMYB97 regulated the salvianolic acid B synthesis as a transcription factor by multiple signaling pathways. In this proposal, the relationship with SmMYB97, SmDELLA1 and SmJAZ8 intend to be further clarified. By elucidating the function of SmMYB97, it was necessary to confirm SmDELLA1 how to regulate the synthesis of salvianolic acid B. In overexpression and knockout transgenic lines of SmMYB97, SmDELLA1 and SmJAZ8, the gene expression pattern and the accumulation of salvianolic acid B were detected and analyzed further, with GA3 and MeJA treatments. The performance was to elucidate the molecular mechanism of SmMYB97 regulating salvianolic acid B biosynthesis under dual signal pathway, and to provide the way for retrieving more transcription factors and enzymes involved in phenolic acids biosynthesis as well as laying a foundation for the production of salvianolic acid B and molecular breeding of high content salvianolic acid B in S. miltiorrhiza.

课题组前期研究发现：丹参中SmMYB97受SmJAZ8调控，通过结合苯丙烷代谢途径SmPAL1和SmTAT1启动子而调控酶基因表达，从而参与丹酚酸B的合成调控；同时SmMYB97与SmDELLA1的存在互作，SmDELLA1已经证实也能够影响总酚酸类的合成；因此推测丹参中存在多信号途径协同作用SmMYB97调控丹酚酸B的合成。本项目拟在前期实验基础上，进一步明确SmMYB97、SmDELLA1和SmJAZ8之间的关系。首先在明确SmMYB97基因功能和SmDELLA1调控丹酚酸B的合成基础上；同时利用SmMYB97、SmDELLA1和SmJAZ8过表达和敲除株系同时检测3条基因对丹酚酸B合成的贡献；并结合GA3和MeJA激素处理，阐明双信号途径下SmMYB97调控丹酚酸B合成的分子机理，为反推更多参与调控丹酚酸合成的转录因子和酶基因提供思路，也为丹参高含量丹酚酸B的分子育种奠定基础。

茉莉酸和赤霉素均能诱导丹参中丹酚酸的积累，但两者之间联系较少。通过前期茉莉酸信号研究发现，我们得到了一个受SmJAZ8负调控的转录因子SmMYB97，通过转基因实验和含量测定等发现，SmMYB97同时正调控丹酚酸和丹参酮类积累，进一步验证发现SmMYB97正调控的靶基因SmPAL1，SmTAT1和SmKSL1，SmCPS1的表达从而影响丹参酮和丹酚酸的积累，而SmJAZ8负调控SmMYB97。另一方面我们研究明确了SmDELLA1同样正向调控丹酚酸的积累，SmDELLA1可以下调SmMYB97对SmPAL1的激活作用，对SmTAT1则是增强其表达，对丹参酮合成基因没有显著变化；同时对赤霉素含量呈现负调控的SmGA2ox11和对赤霉素含量发挥正调控作用的SmK0基因影响极为显著。SmDELLA1与SmJAZ8，SmMYB97同样互作，且体外结果SmDELLA1与SmJAZ8互作较弱，而BIFC实验确定两者显著互作；意味SmMYB97可能是SmDELLA1与SmJAZ8的一个介导靶点，同时分流分别调控茉莉酸和赤霉素途径中影响丹酚酸的积累。本研究系统阐明了SmMYB97通过 JAZ8/DELLA1 调控对酚酸类化合物分子机制，同时结合茉莉酸和赤霉素调控途径探究和解析了更多的影响调控次生代谢的重要酶基因，为后期丹参次生代谢研究提供了理论依据。