生物技术通报 ›› 2022, Vol. 38 ›› Issue (5): 74-83.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0981

• 研究报告 • 上一篇    下一篇

R2R3-MYB转录因子PnMYB1调控三七皂苷生物合成

雷君1,2(), 陈勤1,2, 邓兵1,2, 张金渝3, 刘迪秋1,2, 崔秀明1,2, 葛锋1,2()   

  1. 1.昆明理工大学生命科学与技术学院,昆明 650500
    2.云南省三七资源可持续利用重点实验室,昆明 650500
    3.云南省农业科学院药用植物研究所,昆明 650205
  • 收稿日期:2021-08-02 出版日期:2022-05-26 发布日期:2022-06-10
  • 作者简介:雷君,女,硕士研究生,研究方向:药用植物生物技术;E-mail: 353597247@qq.com
  • 基金资助:
    国家自然科学基金项目(82060692);云南省重大科技计划项目(2019ZF011-2)

Biosynthesis of Panax notoginseng Saponins Regulated by R2R3-MYB Transcription Factor PnMYB1

LEI Jun1,2(), CHEN Qin1,2, DENG Bing1,2, ZHANG Jin-yu3, LIU Di-qiu1,2, CUI Xiu-ming1,2, GE Feng1,2()   

  1. 1. Faculty of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500
    2. Yunnan Key Laboratory of Panax notoginseng Resources Sustainable Development and Utilization,Kunming 650500
    3. Institute of Medicinal Plant,Yunnan Academy of Agricultural Sciences,Kunming 650205
  • Received:2021-08-02 Published:2022-05-26 Online:2022-06-10

摘要:

旨在明确PnMYB1转录因子对三七皂苷生物合成具有调控作用。利用RACE技术获得PnMYB1基因全长,对PnMYB1进行系统发育树分析;构建PnMYB1植物过表达载体并侵染三七细胞,检测转基因三七细胞中人参皂苷R1、Rg1、Re、Rb1和Rd的含量;将PnMYB1与鲨烯合酶(PnSS)、鲨烯环氧化酶(PnSE)、达玛烯二醇合成酶(PnDS)和环阿屯醇合成酶(PnCAS)等参与三七皂苷生物合成途径的关键酶的基因启动子共转染烟草叶片,进行瞬时表达分析,利用GUS表达系统验证PnMYB1转录因子能否与三七皂苷生物合成关键酶基因的启动子相互作用。结果显示,PnMYB1转录因子属于R2R3-MYB家族;在过表达PnMYB1的三七细胞中,五种重要三萜皂苷在转基因细胞中均有不同程度的增加,进一步分析证明PnMYB1转录因子通过激活PnSEPnDS的启动子,促使PnSEPnDS的表达水平显著升高,进而实现对三七皂苷生物合成的调控。PnMYB1转录因子可以同时调控三七皂苷生物合成途径中两个关键酶基因的表达,从而影响三七皂苷的生物合成。

关键词: 三七, 皂苷, 转录因子, 三萜类化合物

Abstract:

This work aims to clarify the regulation role of transcription factor PnMYB1 to Panax notoginseng saponins(PNS)biosynthesis. RACE technique was applied to obtain the full length of PnMYB1 gene,the phylogenetic tree analysis of PnMYB1 were carried out. The plant overexpression vector of PnMYB1 was constructed and infected P. notoginseng cells,and the contents of ginsenosides R1,Rg1,Re,Rb1 and Rd in the transgenic P. notoginseng cells were detected. For instant expression analysis,tobacco leaves were co-transfected with PnMYB1 and the gene promoters of some key enzymes involved in the biosynthetic pathway of PNS,including squalene synthase(PnSS),squalene epoxidase(PnSE),dammarenediol synthase(PnDS)and cycloartenol synthase(PnCAS). The GUS expression system was employed to confirm whether PnMYB1 transcription factor interacted with the promoters of key enzyme genes in the biosynthetic pathway of PNS. The results showed that PnMYB1 transcription factors belonged to the R2R3-MYB family. In PnMYB1-overexpressed cells of P. notoginseng,5 important triterpenoid saponins increased to some extent in the transgenic cells. Further analysis confirmed that PnMYB1 significantly increased the expressions of PnSE and PnDS by activating the promoters of PnSE and PnDS,thus regulating the biosynthesis of PNS. In conclusion,PnMYB1 transcription factor may simultaneously regulate the expressions of two key enzyme genes involved in the biosynthesis of PNS and then affect its biosynthesis.

Key words: Panax notoginseng, saponins, transcription factor, triterpenoid