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Purple Chromoprotein Gene Serves as a New Selection Marker for Transgenesis of the Microalga Nannochloropsis oculata

Among the methods used to screen transgenic microalgae, antibiotics selection has raised environmental and food safety concerns, while the observation of fluorescence proteins could be influenced by the endogenous fluorescence of host chloroplasts. As an alternative, this study isolated the purple c...

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Autores principales: Shih, Chen-Han, Chen, Hsiao-Yin, Lee, Hung-Chieh, Tsai, Huai-Jen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368691/
https://www.ncbi.nlm.nih.gov/pubmed/25793255
http://dx.doi.org/10.1371/journal.pone.0120780
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author Shih, Chen-Han
Chen, Hsiao-Yin
Lee, Hung-Chieh
Tsai, Huai-Jen
author_facet Shih, Chen-Han
Chen, Hsiao-Yin
Lee, Hung-Chieh
Tsai, Huai-Jen
author_sort Shih, Chen-Han
collection PubMed
description Among the methods used to screen transgenic microalgae, antibiotics selection has raised environmental and food safety concerns, while the observation of fluorescence proteins could be influenced by the endogenous fluorescence of host chloroplasts. As an alternative, this study isolated the purple chromoprotein (CP) from Stichodacyla haddoni (shCP). A plasmid in which shCP cDNA is driven by a heat-inducible promoter was linearized and electroporated into 2.5×10(8) protoplasts of Nannochloropsis oculata. Following regeneration and cultivation on an f/2 medium plate for two weeks, we observed 26 colonies that displayed a slightly dark green coloration. After individually subculturing and performing five hours of heat shock at 42°C, a dark brown color was mosaically displayed in five of these colonies, indicating that both untransformed and transformed cells were mixed together in each colony. To obtain a uniform expression of shCP throughout the whole colony, we continuously isolated each transformed cell that exhibited brown coloration and subcultured it on a fresh plate, resulting in the generation of five transgenic lines of N. oculata which stably harbored the shCP gene for at least 22 months, as confirmed by PCR detection and observation by the naked eye. As shown by Western blot, exogenous shCP protein was expressed in these transgenic microalgae. Since shCP protein is biodegradable and originates from a marine organism, both environmental and food safety concerns have been eliminated, making this novel shCP reporter gene a simple, but effective and ecologically safe, marker for screening and isolating transgenic microalgae.
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spelling pubmed-43686912015-03-27 Purple Chromoprotein Gene Serves as a New Selection Marker for Transgenesis of the Microalga Nannochloropsis oculata Shih, Chen-Han Chen, Hsiao-Yin Lee, Hung-Chieh Tsai, Huai-Jen PLoS One Research Article Among the methods used to screen transgenic microalgae, antibiotics selection has raised environmental and food safety concerns, while the observation of fluorescence proteins could be influenced by the endogenous fluorescence of host chloroplasts. As an alternative, this study isolated the purple chromoprotein (CP) from Stichodacyla haddoni (shCP). A plasmid in which shCP cDNA is driven by a heat-inducible promoter was linearized and electroporated into 2.5×10(8) protoplasts of Nannochloropsis oculata. Following regeneration and cultivation on an f/2 medium plate for two weeks, we observed 26 colonies that displayed a slightly dark green coloration. After individually subculturing and performing five hours of heat shock at 42°C, a dark brown color was mosaically displayed in five of these colonies, indicating that both untransformed and transformed cells were mixed together in each colony. To obtain a uniform expression of shCP throughout the whole colony, we continuously isolated each transformed cell that exhibited brown coloration and subcultured it on a fresh plate, resulting in the generation of five transgenic lines of N. oculata which stably harbored the shCP gene for at least 22 months, as confirmed by PCR detection and observation by the naked eye. As shown by Western blot, exogenous shCP protein was expressed in these transgenic microalgae. Since shCP protein is biodegradable and originates from a marine organism, both environmental and food safety concerns have been eliminated, making this novel shCP reporter gene a simple, but effective and ecologically safe, marker for screening and isolating transgenic microalgae. Public Library of Science 2015-03-20 /pmc/articles/PMC4368691/ /pubmed/25793255 http://dx.doi.org/10.1371/journal.pone.0120780 Text en © 2015 Shih et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Shih, Chen-Han
Chen, Hsiao-Yin
Lee, Hung-Chieh
Tsai, Huai-Jen
Purple Chromoprotein Gene Serves as a New Selection Marker for Transgenesis of the Microalga Nannochloropsis oculata
title Purple Chromoprotein Gene Serves as a New Selection Marker for Transgenesis of the Microalga Nannochloropsis oculata
title_full Purple Chromoprotein Gene Serves as a New Selection Marker for Transgenesis of the Microalga Nannochloropsis oculata
title_fullStr Purple Chromoprotein Gene Serves as a New Selection Marker for Transgenesis of the Microalga Nannochloropsis oculata
title_full_unstemmed Purple Chromoprotein Gene Serves as a New Selection Marker for Transgenesis of the Microalga Nannochloropsis oculata
title_short Purple Chromoprotein Gene Serves as a New Selection Marker for Transgenesis of the Microalga Nannochloropsis oculata
title_sort purple chromoprotein gene serves as a new selection marker for transgenesis of the microalga nannochloropsis oculata
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368691/
https://www.ncbi.nlm.nih.gov/pubmed/25793255
http://dx.doi.org/10.1371/journal.pone.0120780
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