Abstract
The glucosylation of 5-position of anthocyanin molecules is one of most important modification step, which is responsible for the stability and color hue of anthocyanin molecules. However, anthocyanin 5-O-glucosyltransferase (5-GT) is unstable and it had been difficult to purify this enzyme by biochemical methods. The isolation of cDNA encoding 5-GT was succeeded first by mRNA differential display (DD) of red and green chemotypes of Perilla frutescens var. crispa. The full-length cDNA was isolated by the screening of perilla cDNA library with cDNA fragments, which was displayed specifically in red perilla. The biological characters of this enzyme were investigated by using the recombinant protein expressed in yeast. We have shown an example of the application of DD technique to the molecular biology in plant secondary metabolisms.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Yamazaki, M. and Saito, K. (2002) Differential display analysis of gene expression in plants. Cell. Mol. Life Sci. 59, 1246–1255.
Yamazaki, M., Gong, Z., Fukuchi-Mizutani, M., et al. (1999) Molecular cloning and biochemical characterization of a novel anthocyanin 5-O-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin. J. Biol. Chem. 274, 7405–7411.
Liang, P. and Pardee, A. (1992) Differential display of eukaryoic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971.
Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Presss, Cold Spring Harbor, NY.
Gong, Z., Yamazaki, M., Sugiyama, M., Tanaka Y., and Saito, K. (1997) Cloning and molecular analysis of structural genes involved in anthocyanin biosynthesis and expressed in form-specific manner in Perilla frutescens. Plant Mol. Biol. 35, 915–927.
Pawlowski, K., Kunze, R., Vries, S.D., and Bisseling T. (1994) Isolation of total poly(A) and polysomal RNA from plant tissues, in Plant Molecular Biology Manual, Gelvin, S.B. and Schilperroort, R.A., eds. Kluwer Academic Publishers, The Netherlands, pp. D5/1–D5/13.
Ashikari, T., Kiuchi-Goto, N., Tanaka, Y., Shibano, Y., Adachi, T. and Yosandzumi, H. (1989) High expression and efficient secretion of Rhizopus oryzae glucoamylase in the yeast Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 30, 515–520.
Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168.
Yamazaki, M., Yamagishi, E, Gong, Z., et al. (2002) Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning, biochemical properties and developmentally regulated expression. Plant Mol. Biol. 48, 401–411.
Acknowledgment
This work was supported, in part, by Grant-in Aid for Scientific Research from the Japan Society for the Promotion of Science, Japan; by Research for the Future Program (00L01605) from the Japan Society for the Promotion of Science.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Yamazaki, M., Saito, K. (2006). Isolation and Characterization of Anthocyanin 5-O-Glucosyltransferase in Perilla frutescens var. crispa by Differential Display. In: Liang, P., Meade, J.D., Pardee, A.B. (eds) Differential Display Methods and Protocols. Methods in Molecular Biology, vol 317. Humana Press. https://doi.org/10.1385/1-59259-968-0:255
Download citation
DOI: https://doi.org/10.1385/1-59259-968-0:255
Publisher Name: Humana Press
Print ISBN: 978-1-58829-338-1
Online ISBN: 978-1-59259-968-4
eBook Packages: Springer Protocols