Establishment of gene expression and silencing assays in Echinacea purpurea

doi:10.1016/j.jia.2025.11.023

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Establishment of gene expression and silencing assays in Echinacea purpurea

¹Medicinal Plant Research Center, College of Life Sciences, South China Agricultural University, Guangzhou, 510642, China

²State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou, 510642, China

Highlights

1. Optimized the experimental parameters for the Agrobacterium tumefaciens-mediated transient expression and the virus-induced gene silencing systems in E. purpurea leaves.

2. Established gene expression and silencing systems in E. purpurea based on the Agrobacterium rhizogenes-mediated cut-dip-budding system.

3. Established an efficient protoplast transient expression system in E. purpurea.

Abstract
References

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摘要

紫锥菊（Echinacea purpurea）是菊科多年生草本园艺植物，富含菊苣酸等次生代谢产物，兼具药用与观赏价值，且栽培适应性较强。然而，紫锥菊次生代谢产物的合成及调控机制尚未明晰。因此，本研究旨在建立紫锥菊基因表达与沉默技术体系，为紫锥菊相关研究提供实验体系。本研究首先实现了农杆菌介导的紫锥菊叶片瞬时表达，经参数优化发现，使用EHA105菌株注射第四片新生叶并培养4天可获得最佳表达效果，可获得最佳外源基因表达效率。随后对已报道的cut-dip-budding（CDB）系统进行改良，建立了发根农杆菌介导的基因表达体系。通过根部感染发根农杆菌，30天后可检测到目的基因的高效表达。利用该体系实现了菊苣酸合成关键基因EpHTT的表达，显著提高了菊苣酸积累量；而通过表达靶向EpHTT的双链RNA，则成功抑制了该基因表达并降低菊苣酸含量。本研究进一步探索了原生质体瞬时表达系统，优化了酶浓度、渗透压等关键参数，成功实现了目的基因在原生质体中的高效瞬时表达。最后，本研究建立了基于烟草脆裂病毒（TRV）介导的紫锥菊基因沉默系统，确定EpCHLH为最佳沉默指示基因，在15天苗龄植株中观察到最优沉默效果。通过沉默菊苣酸合成通路相关基因（EpHCT、EpHTT和EpCAS），有效降低了菊苣酸积累。总之，本研究成功构建了紫锥菊多套基因表达与沉默技术体系，为后续功能研究提供了重要实验体系。

Abstract

Echinacea purpurea (E. purpurea) is a perennial herb and horticulture plant belonging to the Asteraceae family. It is an easy cultivating plant that is well-known for its medicinal (e.g. chicoric acid) and high ornamental value. However, information based on the synthesis and regulatory mechanisms its secondary metabolites are limited. Therefore, to improve research progress on E. purpurea, this study aims to establish gene expression and silencing systems in E. purpurea. First, a transient gene expression system mediated by Agrobacterium tumefaciens was developed in E. purpurea leaves. Following optimization, it was determined that injecting the fourth newly emerged leaf with the EHA105 strain and maintaining it for 4 d yielded the best expression results. Then a previously reported cut-dip-budding (CDB) system was adapted and improved to establish a gene expression system based on Agrobacterium rhizogenes. By infecting the roots with A. rhizogenes, we detected efficient expression of the target gene after 30 d. Using this system, we achieved expression of EpHTT, a key gene involved in chicoric acid synthesis, and significantly increased the accumulation of chicoric acid. By expressing a double-strand RNA targeting EpHTT, we successfully silenced the EpHTT expression and reduced chicoric acid accumulation. We further investigated the protoplast-based transient gene expression system, studied key parameters, such as enzyme concentration and osmotic pressure, and successfully achieved transient expression of the target gene in protoplasts. Finally, a gene-silencing system in E. purpurea mediated by the tobacco rattle virus (TRV) was established and EpCHLH was identified as the optimal silencing indicator gene, with the best silencing effect observed in 15-day-old seedlings. By silencing genes involved in the chicoric acid synthesis pathway, such as EpHCT, EpHTT, and EpCAS, chicoric acid accumulation was successfully reduced. In summary, this study successfully established various gene expression and silencing systems for E. purpurea and providing a valuable toolkit for further functional studies.

Keywords:

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Online: 15 November 2025

Fund:

This work was funded by the National Natural Science Foundation of China (32270381, 32370383), Natural Science Foundation of Guangdong Province (2024B1515020118), the Open Competition Program of Top Ten Critical Priorities of Agricultural Science and Technology Innovation for the 14^th Five-Year Plan of Guangdong Province (2022SDZG07), and Double First-class Discipline Promotion Project (2021B10564001)

About author: #Correspondence Mei Bai, E-mail: baimei924@scau.edu.cn; Hong Wu, E-mail: wh@scau.edu.cn; Xiangxiu Liang, E-mail: liangxiangxiu@scau.edu.cn

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Jianbin Yu, Congyu Wang, Qiao Yang, Zhenpeng Huang, Miaoxin Li, Lijing Gong, Yanqun Li, Mei Bai, Hong Wu, Xiangxiu Liang. 2025.

Establishment of gene expression and silencing assays in Echinacea purpurea . Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.11.023

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