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Efficient heterologous transformation of Chlamydomonas reinhardtii npq2 mutant with the zeaxanthin epoxidase gene isolated and characterized from Chlorella zofingiensis.

Couso I, Cordero BF, Vargas MÁ, Rodríguez H - Mar Drugs (2012)

Bottom Line: The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2.The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm).These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Américo Vespucio no. 49, 41092-Seville, Spain. inmaculada.couso@ibvf.csic.es

ABSTRACT
In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

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Related in: MedlinePlus

Southern blot analysis of genomic DNA from C. zofingiensis. gDNA was digested with ScaI (Lane 1), NdeI (Lane 2), BamHI (Lane 3) or NcoI (Lane 4), electrophoretically separated on a 0.8% agarose gel, blotted and hybridized at high stringency with a probe of 662 bp of the Czzep gene amplified by PCR.
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marinedrugs-10-01955-f002: Southern blot analysis of genomic DNA from C. zofingiensis. gDNA was digested with ScaI (Lane 1), NdeI (Lane 2), BamHI (Lane 3) or NcoI (Lane 4), electrophoretically separated on a 0.8% agarose gel, blotted and hybridized at high stringency with a probe of 662 bp of the Czzep gene amplified by PCR.

Mentions: For the determination of the copy number of the zep gene in the genome of C. zofingiensis, genomic DNA was digested with four different restriction enzymes ScaI, NdeI, BamHI and NcoI and subjected to Southern blot analysis at different conditions of stringency. Using a 662 bp fragment of Czzep as a probe, strong hybridization signals were obtained with the different digestions. The digestion with NcoI and BamHI enzymes, which cut once inside the probe sequence, showed two bands, while digestion with NdeI or ScaI, with no restriction site in the probe, exhibited only one band (Figure 2). These results suggest the presence of a single copy of the zep gene in the genome of C. zofingiensis.


Efficient heterologous transformation of Chlamydomonas reinhardtii npq2 mutant with the zeaxanthin epoxidase gene isolated and characterized from Chlorella zofingiensis.

Couso I, Cordero BF, Vargas MÁ, Rodríguez H - Mar Drugs (2012)

Southern blot analysis of genomic DNA from C. zofingiensis. gDNA was digested with ScaI (Lane 1), NdeI (Lane 2), BamHI (Lane 3) or NcoI (Lane 4), electrophoretically separated on a 0.8% agarose gel, blotted and hybridized at high stringency with a probe of 662 bp of the Czzep gene amplified by PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475266&req=5

marinedrugs-10-01955-f002: Southern blot analysis of genomic DNA from C. zofingiensis. gDNA was digested with ScaI (Lane 1), NdeI (Lane 2), BamHI (Lane 3) or NcoI (Lane 4), electrophoretically separated on a 0.8% agarose gel, blotted and hybridized at high stringency with a probe of 662 bp of the Czzep gene amplified by PCR.
Mentions: For the determination of the copy number of the zep gene in the genome of C. zofingiensis, genomic DNA was digested with four different restriction enzymes ScaI, NdeI, BamHI and NcoI and subjected to Southern blot analysis at different conditions of stringency. Using a 662 bp fragment of Czzep as a probe, strong hybridization signals were obtained with the different digestions. The digestion with NcoI and BamHI enzymes, which cut once inside the probe sequence, showed two bands, while digestion with NdeI or ScaI, with no restriction site in the probe, exhibited only one band (Figure 2). These results suggest the presence of a single copy of the zep gene in the genome of C. zofingiensis.

Bottom Line: The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2.The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm).These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Américo Vespucio no. 49, 41092-Seville, Spain. inmaculada.couso@ibvf.csic.es

ABSTRACT
In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

Show MeSH
Related in: MedlinePlus