丹参miR828和miR858参与次生代谢调控的分子机制

Salvia miltiorrhiza Bunge is a well-known material of traditional Chinese medicine (TCM). It is also an emerging model plant for TCM studies. The genome of S. miltiorrhiza has been preliminarily decoded and significant research progress has been made in key enzyme genes involved in the biosynthesis of secondary metabolites, such as tanshinones and phenolic acids; however, the regulatory mechanism of secondary metabolism is largely unknown. Based on our results from the studies on secondary metabolite biosynthesis-related genes, miRNAs and MYB transcription factors, we assume that miR828 and miR858 play significant regulatory roles in the biosynthesis of secondary metabolites in S. miltiorrhiza. With the aim to test this hypothesis, we propose to construct miR828 and miR858 overexpression vectors and then introduce them into S. miltiorrhiza. The content of main secondary metabolites in transgenics and non-transgenics will be analyzed comparatively. High-throughput degradome sequencing and RNA-seq will be performed for transcriptome-wide analysis of genes directly cleaved or indirectly regulated by miR828 and miR858. All of the data obtained will be further comprehensively analyzed for elucidating the relationships among miR828, miR858, protein-coding genes, and the production of main secondary metabolites in S. miltiorrhiza. We expect that the regulatory network of miR828 and miR858 in secondary metabolism will be constructed and the regulatory mechanism will be finally elucidated. The results will provide useful information for understanding the genetic regulatory mechanism in the biosynthesis of S. miltiorrhiza bioactive compounds and will serve as a basis for development of new S. miltiorrhiza lines with high bioactive compound content through genetic engineering approaches.

丹参是传统中药材，正成为模式药用植物。丹参全基因组序列测定已基本完成，次生代谢产物丹参酮和酚酸等合成途径的关键酶基因研究也已取得进展，但其分子调控机理还很不清楚。基于前期对丹参次生代谢产物生物合成相关基因、miRNA和MYB转录因子的研究结果，我们认为miR828和miR858是丹参次生代谢的重要调控因子。为了验证这一假设，本项目将构建miR828和miR858过表达载体，转化丹参，比较分析转基因和非转基因丹参主要次生代谢产物含量的变化，高通量分析丹参降解组和转录组数字表达谱，从组学水平找出受miR828和miR858直接调控或间接影响的基因，揭示miR828和miR858与蛋白质编码基因和主要次生代谢产物之间的关系，构建miR828和miR858的调控网络并探讨其作用机制，为彻底揭示丹参药用活性成分生物合成的遗传控制机制和利用基因工程手段获得药用活性成分含量高的新品系奠定基础。

丹参是常用大宗中药材，也是基因组序列已经解码的药用模式植物。丹参含脂溶性二萜醌类化合物丹参酮、水溶性的酚酸类化合物丹酚酸以及黄酮类、醌类、三萜类、甾醇等其他成分，揭示次生代谢的调控机理对丹参品种改良具有重要意义。植物微小RNA是一类长度约为21-24个碱基、在植物体内发挥重要调控作用的非蛋白质编码RNA。本项目在克隆丹参MIR828基因的基础上，基于MIR828基因序列构建miR828过表达载体并基于人工miRNA技术构建miR858过表达载体，对农杆菌介导的丹参遗传转化体系进行了优化和改进，完成了农杆菌介导的miR828和miR858过表达载体的丹参遗传转化，基于LC-MS和RNA-seq技术分析了转基因丹参的次生代谢产物和转录组，解析了调控网络相关的家族基因。经上述研究，建立了一个高效的丹参遗传转化体系，发现丹参miR828介导MYB和ta-siRNA调控黄酮类化合物的生物合成，丹参miR858介导MYB基因调控丹参酚酸类化合物的生物合成，此外还系统鉴定出326条丹参次生代谢相关基因和5446条mlncRNA。已在SCI期刊发表论文10篇，最高SCI影响因子6.825，参编专著2章节，申请专利1项；参加了2个国际学术会议；培养博士后3名，博士研究生7名。经过项目组全体成员4年的努力，完成了计划的研究内容，并在此基础上增加了部分与本项目有关的研究工作，达到并部分超过了“验证miR828和miR858是丹参次生代谢的重要调控因子，阐明miR828和miR858作用的分子机制”这一预期目标，取得较好的研究结果，为进一步解析丹参次生代谢产物的合成和调控机制奠定了良好基础，具有较大的科学意义和潜在的应用前景。