Abstract
RNAi (RNA interference, RNA silencing) is a powerful tool in functional genomics. We report here the use of transient RNAi to isolate regulatory factor genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica protoplasts. Double-stranded (ds) RNAs prepared against candidate regulatory factors, which were predicted from an EST library, were introduced into C. japonica protoplasts by polyethylene glycol (PEG)-mediated transformation, and their effects were monitored by real-time reverse transcription (RT)-polymerase chain reaction (PCR). The potential of this transient RNAi system to characterize the functions of regulatory factor genes in alkaloid research is discussed.
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Abbreviations
- MES:
-
2-morpholinoethanesulfonic acid;
- PEG:
-
polyethylene glycol;
- RNAi:
-
RNA interference;
- RT:
-
reverse transcription;
- TF:
-
transcription factor.
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Acknowledgements
We thank Dr. Kentaro Ifuku for his critical reading of this manuscript. This work was supported in part by a grant from the Ministry of Education, Culture, Sports, and Science and Technology of Japan (21248013 to F. S.).
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Yamada, Y., Kato, N., Kokabu, Y., Luo, Q., Dubouzet, J.G., Sato, F. (2010). Identification of Regulatory Protein Genes Involved in Alkaloid Biosynthesis Using a Transient RNAi System. In: Fett-Neto, A. (eds) Plant Secondary Metabolism Engineering. Methods in Molecular Biology, vol 643. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-723-5_3
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DOI: https://doi.org/10.1007/978-1-60761-723-5_3
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