SPL7-miR408-LACs模块调控丹酚酸B生物合成的分子机制

Salvianolic acid B (SalB) is one of the main active components of Salvia miltiorrhiza. The biosynthesis and regulation of SalB are the hot spots. Our previous studies demonstrated that miR408 negatively regulates the biosynthesis of SalB in S. miltiorrhiza, but the molecular mechanism is still unknown. Further research showed that miR408 is regulated by SQUAMOSA promoter binding protein-likes (SPL) transcription factor SPL7 and miR408 targets the transcripts of a few members of laccase (LAC) gene family in S. miltiorrhiza. We put forward a hypothesis that the SPL7-miR408-LACs module regulates the biosynthesis of SalB. LACs probably catalyze RAs into SalB or indirectly regulate the accumulation of SalB. In this proposal, we will further confirm the relation between miR408 and SPL7/LACs by experiments. The cleavage sites of miR408 on LACs mRNA will be validated through RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) method. Whether SPL7 directly binds to the promoter of MIR408 will be verified by yeast one hybrid. We will focus on the function of LAC6/38/43, the target of miR408. We plan to assay the enzyme activity using the yeast in vivo system, study their kinetics to find their natural substrates. We will obtain a serious transgenic S. miltiorrhiza lines, confirm contributions of the enzymes to the accumulation of SalB through transgenic S. miltiorrhiza lines, in which SPL7, miR408 and LACs is overexpressed or knock-down by RNA interference. The molecular mechanism for SPL7-miR408-LAC module in regulating SalB biosynthesis will be elucidated through integrated analysis of transcriptome and metabolome between the transgenic lines and the control. Success of the proposal will increase our knowledge about the biosynthesis and regulation of SalB and lay the foundations for the production of SalB and molecular breeding of high content SalB in S. miltiorrhiza.

丹酚酸B是丹参的主要药效成分之一，其在丹参中的合成与调控是目前研究的热点。课题组前期研究发现，丹参中miR408负调控丹酚酸B的合成，但分子机制尚不清楚。进一步研究表明，miR408的表达受SPL7的调控，且miR408可靶标多个漆酶（LAC）基因家族成员。因此，我们推测丹参中存在SPL7-miR408-LACs模块调控丹酚酸B的合成。本项目拟通过酵母单杂交、RLM-RACE等技术明确SPL7、miR408和LACs之间的作用关系；通过酵母体内酶活检测体系检验LACs的活性，利用酶促反应动力学实验检测其催化底物，探明LAC是否直接催化迷迭香酸聚合形成丹酚酸B；获得SPL7、miR408、LACs等一系列转基因丹参株系，对其进行代谢组和转录组比较分析，阐明SPL7-miR408-LACs模块调控丹酚酸B合成的分子机理，为丹酚酸B的生产和高含量丹酚酸B的分子育种奠定基础。

丹参被誉为“药用模式植物”。丹酚酸B是丹参中的主要药效成分之一，历年来在《中华人民共和国药典》中被作为丹参药材质量控制的指标性成分。提高丹酚酸B的含量是丹参育种工作者长期追求的主要目标之一，同时也是服务于我国中医药产业发展的需要。项目组推测丹参中存在SmSPL7-miR408-SmLACs模块调控丹酚酸B的合成。基于此，本项目通过一系列实验明确了SmSPL7、miR408和SmLACs之间的作用关系，利用酵母单杂交、启动子瞬时激活、SmSPL7过表达和干涉转基因丹参株系中miR408的表达量变化等明确了miR408为SmSPL7的直接靶标；以miR408过表达和干涉转基因丹参株系为材料，检测miR408的预测靶基因LACs的表达变化，并利用5'-RLM-RACE证实SmLAC3基因是miR408的真实靶标；研究证实，SmSPL7抑制丹酚酸B的合成，miR408负调控丹酚酸B的合成；进一步开展了SmLAC3和SmLAC25的功能研究，与预期相吻合，SmLAC3过表达转基因株系中迷迭香酸和丹酚酸B的含量显著提高，木质素的含量较对照株系有所减少；而SmLAC25过表达株系中迷迭香酸和丹酚酸B的含量显著降低，木质素含量显著增加，体外酶活实验表明：重组的SmLAC25蛋白可以直接氧化G-型木质素单体和S-型木质素单体，说明漆酶家族成员具有明显的底物特异性。本项目首次在丹参中鉴定到在转录后水平调控酚酸类物质合成的miRNA（miR408），研究结果证实了丹参中存在调控丹酚酸B合成的SmSPL7-miR408-SmLAC3通路，为今后利用合成生物学生产丹酚酸B以及通过代谢工程提高丹参中丹酚酸B的含量奠定了基础。