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Production of recombinant miraculin protein in carrot callus via Agrobacterium-mediated transformation

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Abstract

Miraculin is a taste-modifying protein that interacts with human sweet-taste receptors and transforms a sour taste into sweet taste. Miraculin is extracted from the miracle fruit (Synsepalum dulcificum). Since mass production of miraculin is difficult because of regional and seasonal limitations, several attempts have been made to express miraculin in various cell systems. In this study, a binary vector containing the miraculin gene under the control of the SWPA2 promotor was introduced into carrot (Daucus carota) callus via Agrobacterium-mediated transformation to synthesize miraculin in carrot cell cultures. After 4 weeks of co-cultivation with Agrobacterium tumefaciens, 20 tentative transgenic callus (TC) lines were obtained on kanamycin selection medium. PCR analysis confirmed that 18 of these 20 lines (TC1–TC18) carried the miraculin gene, and 4 TC lines with high cell growth and gene expression (determined by RT-qPCR) were selected for further analysis. Protein analysis of these four TC lines by SDS-PAGE and Western blot showed that the miraculin protein was stably produced in TC lines. The cell growth showed no correlation with gene expression levels. The DNA content and G1 phase ratio were negatively correlated, whereas the S and G2/M phase ratios were positively correlated with gene expression. The ratio of cell cycle was determined by counting the number of cells in each step through flow cytometric analysis. These results indicate that gene expression was higher in TC lines with active cell division. Overall, our results demonstrate the feasibility of mass production of recombinant miraculin protein in transgenic cell culture systems.

Key message

This study expressed the miraculin gene in carrot callus via Agrobacterium-mediated transformation based on miraculin expression and cell cycle analyses. Our results demonstrate the feasibility of mass production of recombinant miraculin protein in transgenic cell culture systems

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Acknowledgements

This work was supported by a grant from the Next Generation BioGreen21 Program (Project No. PJ013689), Rural Development Administration, Republic of Korea.

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Authors and Affiliations

  1. Division of Animal, Horticultural and Food Sciences, Department of Horticulture, Chungbuk National University, Cheongju, 28644, Republic of Korea

    Yun-Ji Park, Jong-Eun Han & So-Young Park

  2. Department of Forest Genetic Resources, National Institute of Forest Science, 39 Onjeong‑ro, Suwon, 16631, Republic of Korea

    Hyoshin Lee

  3. Korea Research Institute of Bioscience and Biotechnology (KIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea

    Ji-Young Lee

  4. Institute for Global Health Innovations, Duy Tan University, Da Nang, 550000, Viet Nam

    Thanh-Tam Ho

  5. Faculty of Pharmacy, Duy Tan University, Da Nang, 550000, Viet Nam

    Thanh-Tam Ho

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  1. Yun-Ji Park
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  2. Jong-Eun Han
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  3. Hyoshin Lee
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  4. Ji-Young Lee
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  5. Thanh-Tam Ho
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Contributions

YJP and JEH contributed to data acquisition and wrote the manuscript. TTH and HL participated in interpreted data and revising for intellectual content. SYP made substantial contributions to the conception and design of the study.

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Correspondence to Thanh-Tam Ho or So-Young Park.

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The authors declare that they have no conflicts of interest.

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Communicated by Sergio J. Ochatt.

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Park, YJ., Han, JE., Lee, H. et al. Production of recombinant miraculin protein in carrot callus via Agrobacterium-mediated transformation. Plant Cell Tiss Organ Cult 145, 615–623 (2021). https://doi.org/10.1007/s11240-021-02032-3

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  • DOI: https://doi.org/10.1007/s11240-021-02032-3

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