WRKY转录因子调控地黄苯乙醇苷生物合成的机制研究

Phenylethanoid glycosides (PhGs) are a class of higher content constituents in Rehmannia glutinosa L., has extensive biological activities including antioxidant, immunoregulation, anti-inflammatory, and hepatoprotection. However, the molecular regulation of WRKY on biosynthesis of PhGs in R. glutinosa has not been thoroughly investigated. Our preliminary study showed that the content of total PhGs were improved after salicylic acid (SA) or methyl jasmonate (MeJA) treated, and several coexpressed WRKY genes with candidate genes encoding the enzymes of PhGs biosynthesis were screened. So we inferred that WRKY maybe the key regulator in GhGs biosynthesis. In this project, we are going to gain full-length transcriptome of R. glutinosa by PacBio sequencing platform in first. Secondly, we will screen candidate WRKY genes involved in PhGs biosynthesis by RNA-seq for hairy roots with SA and MeJA treatments. Thirdly, we will research the molecular functions of candidate WRKYs by CRISPR/Cas9 method. And the last, we’ll test the regulatory network of WRKY on PhGs biosynthesis using GUS activity analysis and yeast single hybrid technology. This project will preliminarily interpret the molecular regulation network of PhGs biosynthesis in R. glutinosa, lay foundations for quality control and breeding of R. glutinosa, enrich and develop science of Chinese medicine resources and molecular pharmacognosy.

苯乙醇苷是地黄中含量较高的一类活性成分，具有抗氧化、免疫调节、抗炎、保肝等药理活性。前期研究发现水杨酸和茉莉酸甲脂能够促进毛蕊花糖苷和总苯乙醇苷的积累，并筛选到与催化酶基因共表达的WRKY转录因子，据此推测WRKY是地黄苯乙醇苷合成的关键调控因子，但WRKY在苯乙醇苷合成中的分子调控机制尚不清楚。本项目拟以地黄“温85-5”品种为材料开展以下研究：①基于PacBio第三代测序技术构建地黄的全长转录组；②运用RNA-seq技术筛选参与苯乙醇苷合成的候选WRKY转录因子；③以CRISPR/Cas9技术获得纯合突变或双等位突变的地黄植株，确定WRKY在地黄苯乙醇苷合成中的功能；④以RNA-seq结合qRT-PCR分析确定WRKY调控的基因，分别以GUS活性共表达分析和酵母单杂交进行调控网络验证，以期阐明WRKY对地黄苯乙醇苷合成的调控作用，为地黄新品种选育和精确栽培、确保药材质量奠定理论基础。

苯乙醇苷是一类在地黄中含量较高的成分，具有抗肿瘤、免疫调节、抗氧化、保护神经系统、改善记忆等药理作用，然而其在地黄体内合成的分子调控机制研究较为薄弱。项目利用Pacific Bio RS II三代测序技术获得了高质量的地黄转录组数据信息，推导了参与异毛蕊花糖苷等6种苯乙醇苷成分的催化酶。鉴定出37个具有完整开放阅读框（ORF）地黄WRKY转录因子基因，分析了其在SA、MeJA和过氧化氢处理下的表达体系及在地黄不同组织中的表达模式，确定了RgWRKY7、RgWRKY23、RgWRKY34、RgWRKY35、RgWRKY37为地黄调控苯乙醇苷的候选转录因子。RgWRKY37过量表达的毛状根中总苯乙醇苷和毛蕊花糖苷的含量显著提高，毛蕊花糖苷的含量较对照提高了74.44-147.11%，说明RgWRKY37是地黄苯乙醇苷合成的正调控转录因子。建立了高效的地黄CRISPR/Cas9基因编辑体系，RgWRKY37基因敲除的地黄株系苯乙醇苷和毛蕊花糖苷的含量降低。双荧光素酶分析发现RgWRKY37可以激活RgPPO启动子活性，可以正向调控RgPPO的表达。项目的实施获得了地黄的全长转录组，揭示了WRKY参与地黄苯乙醇苷合成的分子调控机制，构建了地黄基因编辑体系，为全面揭示地黄苯乙醇苷合成的分子调控网络奠定基础。