Data_Sheet_1_Functional analysis of Pogostemon cablin farnesyl pyrophosphate synthase gene and its binding transcription factor PcWRKY44 in regulating biosynthesis of patchouli alcohol.PDF

Farnesyl pyrophosphate synthase (FPPS) plays an important role in the synthesis of plant secondary metabolites, but its function and molecular regulation mechanism remain unclear in Pogostemon cablin. In this study, the full-length cDNA of the FPP synthase gene from P. cablin (PcFPPS) was cloned and characterized. The expressions of PcFPPS are different among different tissues (highly in P. cablin flowers). Subcellular localization analysis in protoplasts indicated that PcFPPS was located in the cytoplasm. PcFPPS functionally complemented the lethal FPPS deletion mutation in yeast CC25. Transient overexpression of PcFPPS in P. cablin leaves accelerated terpene biosynthesis, with an ~47% increase in patchouli alcohol. Heterologous overexpression of PcFPPS in tobacco plants was achieved, and it was found that the FPP enzyme activity was significantly up-regulated in transgenic tobacco by ELISA analysis. In addition, more terpenoid metabolites, including stigmasterol, phytol, and neophytadiene were detected compared with control by GC-MS analysis. Furthermore, with dual-LUC assay and yeast one-hybrid screening, we found 220 bp promoter of PcFPPS can be bound by the nuclear-localized transcription factor PcWRKY44. Overexpression of PcWRKY44 in P. cablin upregulated the expression levels of PcFPPS and patchoulol synthase gene (PcPTS), and then promote the biosynthesis of patchouli alcohol. Taken together, these results strongly suggest the PcFPPS and its binding transcription factor PcWRKY44 play an essential role in regulating the biosynthesis of patchouli alcohol.

法尼基焦磷酸合酶(Farnesyl pyrophosphate synthase, FPPS)在植物次生代谢产物合成中发挥重要作用，但广藿香(Pogostemon cablin)中该基因的功能及分子调控机制仍不明确。本研究克隆并鉴定了广藿香来源的FPP合酶基因全长cDNA（命名为PcFPPS）。PcFPPS在不同组织中的表达存在显著差异，在广藿香花中表达量最高。通过原生质体进行的亚细胞定位分析显示，PcFPPS定位于细胞质中。功能互补实验表明，PcFPPS可互补酵母CC25中FPPS基因缺失引发的致死突变。在广藿香叶片中瞬时过表达PcFPPS可促进萜类生物合成，使广藿香醇含量提升约47%。本研究实现了PcFPPS在烟草中的异源过表达，经酶联免疫吸附测定(Enzyme-Linked Immunosorbent Assay, ELISA)分析发现，转基因烟草的FPP酶活性显著上调。此外，通过气相色谱-质谱联用(Gas Chromatography-Mass Spectrometry, GC-MS)分析，相较于对照组，转基因植株中可检测到更多萜类代谢产物，包括豆甾醇、植醇与新植二烯。进一步通过双荧光素酶报告基因测定(Dual-Luciferase Reporter Assay, dual-LUC assay)与酵母单杂交(Yeast One-Hybrid, Y1H)筛选，本研究发现PcFPPS的220 bp启动子可与核定位转录因子PcWRKY44相结合。在广藿香中过表达PcWRKY44可上调PcFPPS及广藿香醇合酶基因(Patchoulol Synthase, PcPTS)的表达水平，进而促进广藿香醇的生物合成。综上，上述研究结果充分表明，PcFPPS及其结合的转录因子PcWRKY44在调控广藿香醇的生物合成过程中发挥关键作用。