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Isolation and characterization of a lycopene ε-cyclase gene of Chlorella (Chromochloris) zofingiensis. Regulation of the carotenogenic pathway by nitrogen and light.

Cordero BF, Couso I, Leon R, Rodriguez H, Vargas MA - Mar Drugs (2012)

Bottom Line: A single copy of Czlcy-e was found in C. zofingiensis.High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene.The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Americo Vespucio, n° 49, Seville 41092, Spain. baldomero@ibvf.csic.es

ABSTRACT
The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

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Southern blot analysis of genomic DNA from C. zofingiensis. (A) DNA was digested with NdeI (lane 1) or PstI (lane 2), electrophoretically separated on a 0.8% agarose gel, blotted and hybridized at 65 °C with a probe of 1194 bp of the lcy-e gene amplified by PCR. A plasmid containing the lcy-e gene was used as a positive control (lane 3). (B) NdeI and PstI restriction sites present in the Czlcy-e gene. The black bar indicates the probe location.
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marinedrugs-10-02069-f003: Southern blot analysis of genomic DNA from C. zofingiensis. (A) DNA was digested with NdeI (lane 1) or PstI (lane 2), electrophoretically separated on a 0.8% agarose gel, blotted and hybridized at 65 °C with a probe of 1194 bp of the lcy-e gene amplified by PCR. A plasmid containing the lcy-e gene was used as a positive control (lane 3). (B) NdeI and PstI restriction sites present in the Czlcy-e gene. The black bar indicates the probe location.

Mentions: To determine the copy number of lcy-e gene in the genome of C. zofingiensis, genomic DNA was digested with two different restriction enzymes (either NdeI or PstI) and subjected to Southern blot analysis at two different conditions of stringency, both in hibridization and washing (42 and 65 °C). Using a 1194-bp fragment of Czlcy-e as a probe, strong hybridization signals were obtained with both digestions. The digestion with PstI enzyme, which cuts once inside the probe sequence, showed two bands, while digestion with NdeI, which cuts at one extreme of the probe, exhibited only one band for the two conditions of stringency tested (Figure 3). These results suggested the presence of a single copy of the lcy-e gene in the genome of C. zofingiensis.


Isolation and characterization of a lycopene ε-cyclase gene of Chlorella (Chromochloris) zofingiensis. Regulation of the carotenogenic pathway by nitrogen and light.

Cordero BF, Couso I, Leon R, Rodriguez H, Vargas MA - Mar Drugs (2012)

Southern blot analysis of genomic DNA from C. zofingiensis. (A) DNA was digested with NdeI (lane 1) or PstI (lane 2), electrophoretically separated on a 0.8% agarose gel, blotted and hybridized at 65 °C with a probe of 1194 bp of the lcy-e gene amplified by PCR. A plasmid containing the lcy-e gene was used as a positive control (lane 3). (B) NdeI and PstI restriction sites present in the Czlcy-e gene. The black bar indicates the probe location.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475274&req=5

marinedrugs-10-02069-f003: Southern blot analysis of genomic DNA from C. zofingiensis. (A) DNA was digested with NdeI (lane 1) or PstI (lane 2), electrophoretically separated on a 0.8% agarose gel, blotted and hybridized at 65 °C with a probe of 1194 bp of the lcy-e gene amplified by PCR. A plasmid containing the lcy-e gene was used as a positive control (lane 3). (B) NdeI and PstI restriction sites present in the Czlcy-e gene. The black bar indicates the probe location.
Mentions: To determine the copy number of lcy-e gene in the genome of C. zofingiensis, genomic DNA was digested with two different restriction enzymes (either NdeI or PstI) and subjected to Southern blot analysis at two different conditions of stringency, both in hibridization and washing (42 and 65 °C). Using a 1194-bp fragment of Czlcy-e as a probe, strong hybridization signals were obtained with both digestions. The digestion with PstI enzyme, which cuts once inside the probe sequence, showed two bands, while digestion with NdeI, which cuts at one extreme of the probe, exhibited only one band for the two conditions of stringency tested (Figure 3). These results suggested the presence of a single copy of the lcy-e gene in the genome of C. zofingiensis.

Bottom Line: A single copy of Czlcy-e was found in C. zofingiensis.High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene.The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Americo Vespucio, n° 49, Seville 41092, Spain. baldomero@ibvf.csic.es

ABSTRACT
The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

Show MeSH
Related in: MedlinePlus