调控三七皂苷生物合成转录因子的克隆与功能研究

Panax notoginseng is a kind of medicinal plant, which belongs to Panax L.. The production of P. notoginseng in Yunnan province occupies most of the total amount in the world. The main active ingredients in P. notoginseng are saponins. The medicines which are mainly composed of saponins of P. notoginseng have become the specific natural medicines in preventing and treating cardiovascular and cerebrovascular diseases. Because of the harsh growth environment, plant diseases and pests, crop rotation needing, low land-capability and difficult to popularize large cultivation of P. notoginseng, the gap between supply and demand has been broadened significantly. The basic research of the biosynthesitic pathway of P. notoginseng saponins needs to be strengthened in order to lay the foundation for solving the medicinal resource shortage by modern biotechnology and ensure the sustainable development of P. notoginseng. In this proposed project, the object is to culture P. notoginseng cells in vitro and metabolic engineering will be used. On the basis of understanding of the key genes in the biosynthesitic pathway of P. notoginseng saponins, methyl jasmonate will be used as inducer and the transcription factors will be obtained by the methods of yeast one-hybrid and homologous cloning. The expressions of key genes in the biosynthesitic pathway of saponins will be studied, which are regulated by the transcription factors. The functions and effects in metabolic flux control of transcription factors will be developed thoroughly. In order to provide theory evidence and technique reference for the high expression system construction (homology or nonhomology) of saponins of P. notoginseng, the advantages of controlling several points by transcription factors need to be performed adequately.

三七为人参属药用植物，全世界绝大部分的三七药材产于云南省。三七皂苷是三七药材的主要活性成分，以三七皂苷为主要成分的药品已成为防治心脑血管疾病的特效天然药物。由于三七种植对生境要求苛刻，病虫害严重，需要轮作，土地利用率低，难以扩大种植面积，导致供需矛盾日渐突出。为保证三七产业的可持续发展，需要借助现代生物技术手段解决药用资源短缺问题。因此，有必要开展与三七皂苷生物合成相关的基础研究工作。本课题以体外培养的三七细胞为研究对象，采用代谢工程研究思路，基于前期对三七皂苷生物合成途径关键基因的认识，以茉莉酸甲酯为诱导剂，采用酵母单杂交和同源法获得影响三七皂苷生物合成的转录因子，开展转录因子对皂苷合成途径关键基因表达的调控研究，明确转录因子在控制皂苷代谢流方面的功能和地位，充分发挥转录因子多点调控优势，为三七皂苷同源或异源高效表达系统的构建提供理论依据和技术参考。

三七(Panax notoginseng)是人参属名贵中药材，其所含皂苷成分是防治心脑血管疾病的特效药。由于三七种植需要轮作，土地利用率低，病虫害严重，导致供需矛盾日渐突出。长远来看，采用现代生物技术是解决药用资源短缺问题的重要手段。因此，有必要开展与三七皂苷生物合成相关的基础研究工作。本研究首先建立三七愈伤组织细胞系，优化其生长条件，并以此作为后续研究材料；利用酵母单杂交技术以及同源克隆法，获得了四个响应茉莉酸甲酯诱导的萜类生物合成相关转录因子基因，分别为PnWRKY1、PnbHLH1、PnMYB1和PnERF1；通过转录因子基因在细胞中的过表达以及相关基因的表达分析，研究了转录因子对三七皂苷合成途径中关键酶基因的表达调控以及功能，成功获得多株分别过表达四种转录因子的细胞系，并且证明转录因子通过调控皂苷合成途径中关键酶基因的表达来间接影响皂苷合成，其调控作用具备“多点调控”的特点；与野生型相比，转基因细胞系的皂苷含量最高增加了一倍；机理研究表明，转录因子能够与三七皂苷合成途径中关键酶基因的启动子序列结合，促进关键酶基因的表达，最终增加了三七皂苷的合成数量。以上研究结果证明了转录因子在三七皂苷合成中具有比途径中关键酶更高的调控地位，其“多点调控”的特性更具优势。项目的开展为三七皂苷同源或异源高效表达系统的构建提供了理论依据和技术参考。