title: Subtissue-specific evaluation of promoter efficiency by quantitative fluorometric assay in laser microdissected tissues of rapeseed.
abstract: β-glucuronidase (GUS) is a useful reporter for the evaluation of promoter characteristics in transgenic plants. Here, we introduce an original technique to quantify the strength of promoters at subtissue resolution of cell clusters. The method combines cryotomy, laser microdissection, and improved fluorometric analysis of GUS activity using 6-chloro-4-methylumbelliferyl-β-D-glucuronide as an efficient fluorogenic substrate for kinetic studies in plants. The laser microdissection/6-chloro-4-methylumbelliferyl-β-D-glucuronide method is robust and reliable in a wide range of GUS expression levels and requires extremely low (few cells) tissue amounts. Suitability of the assay was demonstrated on rapeseed (Brassica napus) plants transformed with a P35S2::GUS construct. GUS expression patterns were visualized and quantified in approximately 30 tissues of vegetative and generative organs. Considerable differences in promoter activity within the tissues are discussed in relation to the cell type and developmental state.
authors.affiliation: Department of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Jasik
authors.forename: Silke
authors.initials: S
authors.lastname: Schiebold
authors.forename: Hardy
authors.initials: H
authors.lastname: Rolletschek
authors.forename: Peter
authors.initials: P
authors.lastname: Denolf
authors.forename: Katrien
authors.initials: K
authors.lastname: Van Adenhove
authors.forename: Thomas
authors.initials: T
authors.lastname: Altmann
authors.forename: Ljudmilla
authors.initials: L
authors.lastname: Borisjuk
mesh_terms: D029688:Brassica napus / Q000235:genetics*; D005470:Fluorometry / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D020719:Glucuronides / Q000032:analysis; D061346:Laser Capture Microdissection / Q000379:methods*; D009928:Organ Specificity / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D011401:Promoter Regions, Genetic
chemical_list: C571110:6-chloro-4-methylumbelliferyl-beta-D-glucuronide; D020719:Glucuronides; D005966:Glucuronidase