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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

Using PCR to detect of the existence of transferred gene in the transformed N. oculata.Plasmid phr-shCP was used to transform N. oculata. After transformation, all microalgae cells were cultured, and the putative clones were selected to determine the existence of shCP fragment in N. oculata transformed with phr-shCP at the expected size of 0.684 kb. The wild-type (WT) strain served as a negative control, whereas plasmid phr-shCP served as a positive control. The small subunit 18S ribosomal RNA (18s) served as the internal control.
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pone.0120780.g003: Using PCR to detect of the existence of transferred gene in the transformed N. oculata.Plasmid phr-shCP was used to transform N. oculata. After transformation, all microalgae cells were cultured, and the putative clones were selected to determine the existence of shCP fragment in N. oculata transformed with phr-shCP at the expected size of 0.684 kb. The wild-type (WT) strain served as a negative control, whereas plasmid phr-shCP served as a positive control. The small subunit 18S ribosomal RNA (18s) served as the internal control.

Mentions: To confirm whether the exogenous shCP cDNA exists in host cells, genomic DNAs were isolated from the 26 subcultured strains of N. oculata which were grown on plates after gene transfer of phr-shCP. PCR detection showed that no DNA fragment had been amplified from the wild-type colony. On the other hand, out of 26 colonies, a 684-bp fragment was amplified from 24, with only CP25 and CP26 failing to yield PCR products (Fig. 3). However, as mentioned above, only five transformants, including CP5, CP10, CP15, CP19 and CP23, presented dark brown coloration, indicating that the exogenous shCP gene in some transformants might have been silenced, resulting in the absence of dark brown coloration, even though the shCP gene had been transferred. Based on the number of transformants expressing shCP, we calculated the expression rate of foreign shCP gene in this study to be around 20%, or five out of 24 PCR-positive colonies.


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)

Using PCR to detect of the existence of transferred gene in the transformed N. oculata.Plasmid phr-shCP was used to transform N. oculata. After transformation, all microalgae cells were cultured, and the putative clones were selected to determine the existence of shCP fragment in N. oculata transformed with phr-shCP at the expected size of 0.684 kb. The wild-type (WT) strain served as a negative control, whereas plasmid phr-shCP served as a positive control. The small subunit 18S ribosomal RNA (18s) served as the internal control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120780.g003: Using PCR to detect of the existence of transferred gene in the transformed N. oculata.Plasmid phr-shCP was used to transform N. oculata. After transformation, all microalgae cells were cultured, and the putative clones were selected to determine the existence of shCP fragment in N. oculata transformed with phr-shCP at the expected size of 0.684 kb. The wild-type (WT) strain served as a negative control, whereas plasmid phr-shCP served as a positive control. The small subunit 18S ribosomal RNA (18s) served as the internal control.
Mentions: To confirm whether the exogenous shCP cDNA exists in host cells, genomic DNAs were isolated from the 26 subcultured strains of N. oculata which were grown on plates after gene transfer of phr-shCP. PCR detection showed that no DNA fragment had been amplified from the wild-type colony. On the other hand, out of 26 colonies, a 684-bp fragment was amplified from 24, with only CP25 and CP26 failing to yield PCR products (Fig. 3). However, as mentioned above, only five transformants, including CP5, CP10, CP15, CP19 and CP23, presented dark brown coloration, indicating that the exogenous shCP gene in some transformants might have been silenced, resulting in the absence of dark brown coloration, even though the shCP gene had been transferred. Based on the number of transformants expressing shCP, we calculated the expression rate of foreign shCP gene in this study to be around 20%, or five out of 24 PCR-positive colonies.

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