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Purple chromoprotein gene serves as a new selection marker for transgenesis of the microalga Nannochloropsis oculata.

Shih CH, Chen HY, Lee HC, Tsai HJ - PLoS ONE (2015)

Bottom Line: Following regeneration and cultivation on an f/2 medium plate for two weeks, we observed 26 colonies that displayed a slightly dark green coloration.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Detection of shCP protein encoded from plasmid phr-shCP in the transgenic line of N. oculata.The polyclonal antibody against shCP was used to perform immunoblot analysis of total soluble proteins extracted from wild-type (WT) algal cells (60 μg), as well as transgenic algal cells of line CP10 (50 μg; transgenic phr-shCP). The recombinant protein shCP (His-tagged shCP; His-shCP) extracted from E. coli served as a positive control. The His-tagged shCP produced by E. coli and the non-His-tagged shCP produced by N. oculata were detected, as indicated by the arrow. Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control.
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pone.0120780.g005: Detection of shCP protein encoded from plasmid phr-shCP in the transgenic line of N. oculata.The polyclonal antibody against shCP was used to perform immunoblot analysis of total soluble proteins extracted from wild-type (WT) algal cells (60 μg), as well as transgenic algal cells of line CP10 (50 μg; transgenic phr-shCP). The recombinant protein shCP (His-tagged shCP; His-shCP) extracted from E. coli served as a positive control. The His-tagged shCP produced by E. coli and the non-His-tagged shCP produced by N. oculata were detected, as indicated by the arrow. Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control.

Mentions: To confirm expression of the recombinant shCP protein in the transgenic microalga, we extracted total soluble proteins from transgenic line CP10 and analyzed them by Western blot using polyclonal antibody against shCP protein. Similar to the recombinant shCP produced by E. coli, which served as a positive control (Fig. 5, lane 3), a positive hybridization band with approximate molecular weight of 26 kDa was detected in the proteins extracted from the algal cells of transgenic strain CP10 (Fig. 5, lane 2). However, no signal recognized by antibody against shCP was shown for the proteins extracted from wild-type algal cells (Fig. 5, lane 1). Even though total protein (60 μg) load on gel from wild-type exceeded that from transgenic strain CP10 (50 μg), as indicated by antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as internal control, no detectable band was shown on the gel. This line of evidence suggested that the heterologous shCP protein was correctly expressed by transformed algal cells derived from the transgenic line CP10 of N. oculata.


Purple chromoprotein gene serves as a new selection marker for transgenesis of the microalga Nannochloropsis oculata.

Shih CH, Chen HY, Lee HC, Tsai HJ - PLoS ONE (2015)

Detection of shCP protein encoded from plasmid phr-shCP in the transgenic line of N. oculata.The polyclonal antibody against shCP was used to perform immunoblot analysis of total soluble proteins extracted from wild-type (WT) algal cells (60 μg), as well as transgenic algal cells of line CP10 (50 μg; transgenic phr-shCP). The recombinant protein shCP (His-tagged shCP; His-shCP) extracted from E. coli served as a positive control. The His-tagged shCP produced by E. coli and the non-His-tagged shCP produced by N. oculata were detected, as indicated by the arrow. Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4368691&req=5

pone.0120780.g005: Detection of shCP protein encoded from plasmid phr-shCP in the transgenic line of N. oculata.The polyclonal antibody against shCP was used to perform immunoblot analysis of total soluble proteins extracted from wild-type (WT) algal cells (60 μg), as well as transgenic algal cells of line CP10 (50 μg; transgenic phr-shCP). The recombinant protein shCP (His-tagged shCP; His-shCP) extracted from E. coli served as a positive control. The His-tagged shCP produced by E. coli and the non-His-tagged shCP produced by N. oculata were detected, as indicated by the arrow. Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control.
Mentions: To confirm expression of the recombinant shCP protein in the transgenic microalga, we extracted total soluble proteins from transgenic line CP10 and analyzed them by Western blot using polyclonal antibody against shCP protein. Similar to the recombinant shCP produced by E. coli, which served as a positive control (Fig. 5, lane 3), a positive hybridization band with approximate molecular weight of 26 kDa was detected in the proteins extracted from the algal cells of transgenic strain CP10 (Fig. 5, lane 2). However, no signal recognized by antibody against shCP was shown for the proteins extracted from wild-type algal cells (Fig. 5, lane 1). Even though total protein (60 μg) load on gel from wild-type exceeded that from transgenic strain CP10 (50 μg), as indicated by antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as internal control, no detectable band was shown on the gel. This line of evidence suggested that the heterologous shCP protein was correctly expressed by transformed algal cells derived from the transgenic line CP10 of N. oculata.

Bottom Line: Following regeneration and cultivation on an f/2 medium plate for two weeks, we observed 26 colonies that displayed a slightly dark green coloration.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus