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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

Czzep scheme. The diagram shows that the Czzep gene consists of six exons (I–VI) and five introns (A–E). The 5′ UTR and 3′ UTR sequences are indicated with arrows and correspond to the positions 1–2 bp and 3669–5687 bp, respectively. Numbers represent the cDNA coordinates (bp). UTR, untranslated region.
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marinedrugs-10-01955-f001: Czzep scheme. The diagram shows that the Czzep gene consists of six exons (I–VI) and five introns (A–E). The 5′ UTR and 3′ UTR sequences are indicated with arrows and correspond to the positions 1–2 bp and 3669–5687 bp, respectively. Numbers represent the cDNA coordinates (bp). UTR, untranslated region.

Mentions: Different degenerate primers were designed on the basis of the conserved motifs present in ZEP amino acid sequences from microalgae and plants. A partial Czzep cDNA fragment of 1100 bp was isolated by PCR amplification using the degenerate primers ZEP-1F and ZEP-1R (Table 1). A complete basic local alignment search tool (BLAST) homology search in the Genbank database (NCBI) showed that this fragment had enough similarity with the ZEP genes from other species, and provided sequence information for designing specific primers for rapid amplification of 5′ and 3′ cDNA ends (RACE-PCR). This analysis generated a full-length cDNA of 3836 bp, which contained an Open Reading Frame (ORF) of 1791 bp, a very short two nucleotides of 5′ untranslated region (UTR), and a long 3′ UTR of 2043 nucleotides. The predicted protein has 596 amino acid residues, with an estimated molecular weight of 64.50 kDa, a theoretical isoelectric point of 8.31 and an instability index of 29.87 (data obtained with ProtParam program). The differences between the C. zofingiensis zep gene and the cDNA sequence were compared and revealed the presence of six exons and five introns (Figure 1).


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)

Czzep scheme. The diagram shows that the Czzep gene consists of six exons (I–VI) and five introns (A–E). The 5′ UTR and 3′ UTR sequences are indicated with arrows and correspond to the positions 1–2 bp and 3669–5687 bp, respectively. Numbers represent the cDNA coordinates (bp). UTR, untranslated region.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

marinedrugs-10-01955-f001: Czzep scheme. The diagram shows that the Czzep gene consists of six exons (I–VI) and five introns (A–E). The 5′ UTR and 3′ UTR sequences are indicated with arrows and correspond to the positions 1–2 bp and 3669–5687 bp, respectively. Numbers represent the cDNA coordinates (bp). UTR, untranslated region.
Mentions: Different degenerate primers were designed on the basis of the conserved motifs present in ZEP amino acid sequences from microalgae and plants. A partial Czzep cDNA fragment of 1100 bp was isolated by PCR amplification using the degenerate primers ZEP-1F and ZEP-1R (Table 1). A complete basic local alignment search tool (BLAST) homology search in the Genbank database (NCBI) showed that this fragment had enough similarity with the ZEP genes from other species, and provided sequence information for designing specific primers for rapid amplification of 5′ and 3′ cDNA ends (RACE-PCR). This analysis generated a full-length cDNA of 3836 bp, which contained an Open Reading Frame (ORF) of 1791 bp, a very short two nucleotides of 5′ untranslated region (UTR), and a long 3′ UTR of 2043 nucleotides. The predicted protein has 596 amino acid residues, with an estimated molecular weight of 64.50 kDa, a theoretical isoelectric point of 8.31 and an instability index of 29.87 (data obtained with ProtParam program). The differences between the C. zofingiensis zep gene and the cDNA sequence were compared and revealed the presence of six exons and five introns (Figure 1).

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