生物技术通报 ›› 2022, Vol. 38 ›› Issue (3): 50-58.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0706

• 研究报告 • 上一篇    下一篇

三七细胞中共超表达FPSSS对皂苷合成的影响

杨延1(), 于龙凤1, 王绍梅1, 李卫娜2, 葛锋3()   

  1. 1.滇西科技师范学院,临沧 677000
    2.西京学院,西安 710000
    3.昆明理工大学,昆明 650500
  • 收稿日期:2021-06-02 出版日期:2022-03-26 发布日期:2022-04-06
  • 作者简介:杨延,女,硕士研究生,研究方向:药用植物生物技术;E-mail: yangyan3650199@163.com
  • 基金资助:
    云南省地方本科高校(部分)基础研究联合专项基金(2019FH001-123)

Effects of FPS and SS Co-overexpression in Panax notoginseng Cells on Saponins Synthesis

YANG Yan1(), YU Long-feng1, WANG Shao-mei1, LI Wei-na2, GE Feng3()   

  1. 1. West Yunnan University,Lincang 677000
    2. Xijing University,Xi’an 710000
    3. Kunming University of Science and Technology,Kunming 650500
  • Received:2021-06-02 Published:2022-03-26 Online:2022-04-06

摘要:

研究三七细胞中共超表达法呢基焦磷酸合酶(farnesyl pyrophasphate synthase,FPS)基因、鲨烯合酶(squalene synthase,SS)基因对三七细胞皂苷合成的影响。利用农杆菌EHA105将pCAMBIA1300S-FPS超表达载体转入已超表达SS的三七细胞内,双抗生素培养及基因组PCR筛选转双基因阳性株系、qRT-PCR测阳性株系中FPSSS表达量,分光光度法及色谱法分别测总皂苷和单体皂苷量。在4株转FPSSS细胞中,FPS的相对表达量均比两对照(未转基因细胞和仅转SS细胞)高,最高分别是未转基因对照的3.26倍和仅转SS对照的3.74倍;SS的相对表达量与仅转SS对照相比无显著差异,但均比转基因对照高;总皂苷含量均比两对照高,最高分别是未转基因对照的2.46倍和仅转SS对照的1.73倍;Re、Rh1、Rd和Rg1 单体皂苷量较两对照大多有不同幅度增加。在三七细胞中共超表达FPSSS,可有效提高细胞中的总皂苷和部分单体皂苷量;此外,与仅超表达SS比较,共超表达FPSSS能够进一步提高皂苷含量。

关键词: 法呢基焦磷酸合酶, 鲨烯合酶, 三七皂苷, 共超表达

Abstract:

This work aims to study the effect of FPS(farnesyl pyrophasphate synthase)and SS(squalene synthase)co-overexpression in Panax notoginseng cells on saponins synthesis. The pCAMBIA1300S-FPS overexpression vector was transferred into the P. notoginseng cells in which SS was overexpressed in advance by Agrobacterium tumefaciens EHA105. Transgenic cell lines were screened through double antibiotic culture and genomic PCR. Relative expressions of FPS and SS in the transgenic cell lines were analyzed by qRT-PCR. Contents of P. notoginseng saponins(PNS)and monomer saponins were detected by spectrophotometry and chromatography,respectively. In four transgenic cell lines with overexpression of both FPS and SS,relative expression levels of FPS were all higher than those of two control groups of the non-transgenic line and the line in which only SS was overexpressed,and the highest level was respectively 3.26 times and 3.74 times of the above two control lines;relative expressions of SS showed no significant difference compared with the line in which only SS was overexpressed,but were higher than the non-transgenic line. Contents of PNS were all higher than those of the non-transgenic line and the line in which only SS was overexpressed,and the highest level was respectively 2.46 times and 1.73 times of the above two control lines. Contents of monomer saponins Re,Rh1,Rd and Rg1 were mostly higher than those of the control lines. Overexpression of FPS and SS in P. notoginseng cells may enhance the accumulation of PNS and monomer saponins. In addition,co-overexpression of FPS and SS may further improve PNS content compared with the overexpression of only SS.

Key words: farnesyl pyrophasphate synthase, squalene synthase, Panax notoginseng saponins, co-overexpression