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Im“plant”ing of Mammalian Glycosyltransferase Gene into Plant Suspension-Cultured Cells Using Agrobacterium-Mediated Transformation

  • Hiroyuki Kajiura
  • Kazuhito Fujiyama
Part of the Methods in Molecular Biology book series (MIMB, volume 1321)

Abstract

Enzymatic activity assay of exogenous glycosyltransferase (GT) and glycosylhydrolase (GH) expressed in plants is an important analysis for determination of the expression of the gene of interest. However, generations and establishment of in planta transgenic lines are time-consuming. Furthermore, the expression levels and the activities of the exogenous GTs and GHs are quite low and weak, the radiolabeled donor substrate had to be used to analyze the enzymatic activity. Here, we describe a protocol for the generation of transgenic plants using suspension-cultured cells and a high sensitive assay for GT, especially β1,4-galactosyltransferase, using microsomal fraction from plant cells and fluorescent-labeled sugar chains as an acceptor substrate. This method enables less-time-consuming preparation of stable transgenic plants, non-radiolabeled, high-throughput detail analysis which includes mass spectrometric analysis and exo-glycosidase digestions.

Key words

Glycosyltransferase assay β1,4-galactosyltransferase Nicotiana tabacum L. cv. Bright Yellow 2 Agrobacterium-mediated gene expression High-performance liquid chromatography PA-sugar chain 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Hiroyuki Kajiura
    • 1
  • Kazuhito Fujiyama
    • 1
  1. 1.The International Center for BiotechnologyOsaka UniversityOsakaJapan

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