Abstract
Reticuline is a key compound in the biosynthetic pathway for isoquinoline alkaloids in plants, which include morphine, codeine and berberine. We established cultured California poppy (Eschscholzia californica) cells, in which berberine bridge enzyme (BBE) was knocked down by RNA interference, to accumulate the important key intermediate reticuline. Both BBE mRNA accumulation and enzyme activity were effectively suppressed in transgenic cells. In these transgenic cells, end-products of isoquinoline alkaloid biosynthesis, such as sanguinarine, were considerably reduced and reticuline was accumulated at a maximum level of 310 μg/g-fresh weight. In addition, 1 g-fresh weight of these cells secreted significant amounts of reticuline into the medium, with a maximum level of 6 mg/20 mL culture medium. These cells also produced a methylated derivative of reticuline, laudanine, which could scarcely be detected in control cells. We discuss the potential application of RNAi technology in metabolic modification and the flexibility of plant secondary metabolism.
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Abbreviations
- BBE:
-
Berberine bridge enzyme
- RNAi:
-
RNA interference
- dsRNA:
-
Double-stranded RNA
- siRNA:
-
Small interfering RNA
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Acknowledgements
We appreciate Dr. P Waterhouse for his kind gifts of pKANNIBAL and pART27 and Mr. T Takemura for his excellent technical assistance in maintaining stock cultures. We thank Dr. W Cui of Kobe Pharmaceutical University and Mitsui Chemicals Inc for their generous gifts of the alkaloids. This research was supported in part by Grants from the Research for the Future Program (JSPS-RFTF00L01606) of the Japan Society for the Promotion of Science (to FS) and the New Energy and Industrial Technology Development Organization for projects in the “Development of Transgenic Plants for Production of Industrial Materials” (to FS).
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Fujii, N., Inui, T., Iwasa, K. et al. Knockdown of berberine bridge enzyme by RNAi accumulates (S)-reticuline and activates a silent pathway in cultured California poppy cells. Transgenic Res 16, 363–375 (2007). https://doi.org/10.1007/s11248-006-9040-4
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DOI: https://doi.org/10.1007/s11248-006-9040-4