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          Institute: MPI für Pflanzenzüchtungsforschung     Collection: Dept. Plant Developmental Biology (George Coupland)     Display Documents



ID: 42405.0, MPI für Pflanzenzüchtungsforschung / Dept. Plant Developmental Biology (George Coupland)
Use of red fluorescent protein from Discosoma sp (dsRED) as a reporter for plant gene expression
Authors:Jach, G.; Binot, E.; Frings, S.; Luxa, K.; Schell, J.
Language:English
Date of Publication (YYYY-MM-DD):2001-11
Title of Journal:Plant Journal
Journal Abbrev.:Plant J.
Volume:28
Issue / Number:4
Start Page:483
End Page:491
Review Status:Peer-review
Audience:Experts Only
Abstract / Description:The suitability of the recently described red fluorescent protein dsRED from reef corals for use as a reporter in plant molecular biology was investigated. Based on the clone pDSRED (Clontech), plant expression vectors were constructed for constitutive dsRED expression in the cytosol, the endoplasmic reticulum and the vacuole. Fluorescence microscopy of tobacco BY2 suspension culture cells transiently expressing the plant vectors generated proved that cytosolic expression of the dsRED gives rise to readily detectable levels of red fluorescence, whereas expression in the ER was poor. Vacuolar dsRED expression did not result in any significant fluorescence. dsRED transgenic tobacco SR1 plants were generated to test the sensitivity of dsRED as a reporter in an autofluorescent background, and to identify the possible impact of the introduced fluorescent protein on morphogenesis, plant development and fertility. During the transformation and regeneration phase plants did not show any abnormalities, indicating that dsRED is not interfering with plant development and morphogenesis. Regenerated plants were analysed by PCR, Western blot and fluorescence microscopy for the presence and expression of the transferred genes. The filter sets chosen for fluorescence microscopy proved to be able to block the red chlorophyll fluorescence completely, allowing specific dsRED detection. Best expression levels were obtained with dsRED targeted to the cytosol or chloroplasts. ER-targeted expression of dsRED also gave rise to readily detectable fluorescence levels, whereas vacuolar expression yielded no fluorescence. dsRED transgenic plant lines expressing the protein in the cytosol, ER or chloroplast proved to be fertile. Seed set and germination were normal, except that the seeds and seedlings maintained the red fluorescence phenotype.
Free Keywords:dsRED; reporter genes; fluorescent proteins; GFP; tobacco; transgenic plants
Comment of the Author/Creator:Date: 2001, NOV
External Publication Status:published
Document Type:Article
Communicated by:N.N.
Affiliations:MPI für Pflanzenzüchtungsforschung
External Affiliations:Max Planck Inst Zuchtungsforsch, Carl von Linne Weg 10, D-50829; Cologne, Germany; Max Planck Inst Zuchtungsforsch, D-50829 Cologne, Germany; Dept Microbiol & Immunol, Cologne, Germany
Identifiers:ISI:000172789200011 [ID No:1]
ISSN:0960-7412 [ID No:2]
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