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Cloning and functional analysis of EpGHQH1 in shikonin production of Echium plantagineum

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Abstract

Shikonins are the main naphthoquinones in the root periderm of Echium plantagineum, as well as the hypocotyl and root hairs of young seedlings. These red pigments can be used as dyes or for medicinal uses since ancient times. Geranylhydroquinone 3″-hydroxylase (GHQH) has recently been identified to catalyze the C-3″ site of geranylhydroquinone to form 3″-hydroxy-geranyl-hydroquinone in shikonin biosynthetic pathway. In this study, we cloned a geranylhydroquinone 3″-hydroxylase candidate gene (EpGHQH1) from E. plantagineum, and further characterized its function in shikonin biosynthesis using the hairy root system by overexpression and RNA interference strategies. The obtained results showed that overexpression of EpGHQH1 increased the accumulation of shikonins, showing 2.1-fold higher in acetylshikonin yield than that of control lines. On the contrary, down-regulation of EpGHQH1 resulted in a decline in shikonin production. Furthermore, the EpGHQH1 expression levels in germinated seedlings showed a high positive relation with shikonin accumulation. These findings demonstrated the function of EpGHQH1 in enhancing shikonin biosynthesis of E. plantagineum, and provided the first example of transgenic hairy root system from E. plantagineum for functional analysis of genes in metabolite biosynthesis.

Key message

We reported a possible GHQH gene (EpGHQH1), and demonstrated its positive role in shikonin biosynthesis of E. plantagineum with the transgenic hairy root system by overexpression and RNAi strategies.

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All data generated or analysed during this study are included in this published article and supplemental materials.

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Acknowledgements

We appreciate Dr. Cheng-Yi Tang for his suggestions and discussion on design of the experiments.

Funding

This research was supported by the National Natural Science Foundation of China (Grant Nos. 31771413, U1903201, 31970321, 31670298), the Open Project Program from MOE Key Laboratory of Molecular Epigenetics of China, the Natural Science Foundation of Jiangsu Bureau of Science and Technology (Grant No. BK20191254), and the Program for Changjiang Scholars and Innovative Research Team in University from the Ministry of Education of China (Grant No. IRT_14R27).

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Author notes

  1. Jiangyan Fu, Guihua Lu, and Minkai Yang contributed equally to this work.

Authors and Affiliations

  1. Institute for Plant Molecular Biology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210023, China

    Jiangyan Fu, Guihua Lu, Minkai Yang, Hua Zhao, Wencai Jie, Aliya Fazal, Xuan Wang, Zhongling Wen, Rongjun Fang, Jinliang Qi & Yonghua Yang

  2. School of Life Sciences, Huaiyin Normal University, Huaian, 223300, China

    Guihua Lu

  3. Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, 130024, China

    Bao Liu

  4. Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, 210037, China

    Guihua Lu, Tongming Yin, Jinliang Qi & Yonghua Yang

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  1. Jiangyan Fu
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Contributions

YHY, JLQ conceived and designed the experiments. JYF, HZ, and MKY performed the experiments. JYF, GHL, and MKY analyzed the data. GHL, WCJ, AF, XW, ZLW, RJF, BL, and TMY contributed to resources. JYF wrote the draft of the manuscript. YHY, JLQ, and GHL contributed to review and edit the manuscript. All authors carefully checked and approved the final manuscript.

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Correspondence to Jinliang Qi or Yonghua Yang.

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Communicated by: Yi Li.

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Fu, J., Lu, G., Yang, M. et al. Cloning and functional analysis of EpGHQH1 in shikonin production of Echium plantagineum. Plant Cell Tiss Organ Cult 144, 533–543 (2021). https://doi.org/10.1007/s11240-020-01976-2

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