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  Use of red fluorescent protein from Discosoma sp (dsRED) as a reporter for plant gene expression

Jach, G., Binot, E., Frings, S., Luxa, K., & Schell, J. (2001). Use of red fluorescent protein from Discosoma sp (dsRED) as a reporter for plant gene expression. Plant Journal, 28(4), 483-491.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0012-3E44-2 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0012-3E45-F
Genre: Journal Article

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 Creators:
Jach, G.1, Author              
Binot, E.1, Author              
Frings, S.1, Author              
Luxa, K.2, Author              
Schell, J.1, Author              
Affiliations:
1Dept. of Genetic Principles of Plant Breeding (Jozef Schell), MPI for Plant Breeding Research, Max Planck Society, ou_1113567              
2Dept. of Plant Developmental Biology (George Coupland), MPI for Plant Breeding Research, Max Planck Society, ou_1113571              

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Free keywords: dsRED; reporter genes; fluorescent proteins; GFP; tobacco; transgenic plants
 Abstract: 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.

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Language(s): eng - English
 Dates: 2001-11
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
 Identifiers: eDoc: 42405
ISI: 000172789200011
 Degree: -

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Title: Plant Journal
  Alternative Title : Plant J.
Source Genre: Journal
 Creator(s):
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Publ. Info: -
Pages: - Volume / Issue: 28 (4) Sequence Number: - Start / End Page: 483 - 491 Identifier: ISSN: 0960-7412