Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii.
For the model organism Chlamydomonas reinhardtii, a codon-adapted gene variant of the extracellular luciferase of Gaussia princeps was generated as a sensitive molecular tool to study gene expression from the nuclear genome. In the past, monitoring promoter activity in Chlamydomonas employing the commonly used luciferase encoded by Renilla reniformis was hampered due to the detection limit of the reporter assay, especially if analyzing weak promoters. In this work, the expression of Gaussia-luciferase from such promoters resulted in an average luminescent activity at least 500 times higher than that detected for the Renilla enzyme. The wildtype signal peptide of Gaussia princeps efficiently mediated the export of the luciferase into the culture medium of Chlamydomonas strain cw15arg ( - ), and the characterization of the secreted protein showed an unexpected temperature instability, probably arising from post-translational modifications made by the algae. To further test the utility of Gaussia-luciferase, promoter sequences originating from different viral genomes were analyzed for their ability to drive transgene expression in Chlamydomonas. Solely, the 35S-promoter of the Cauliflower mosaic virus (CaMV) displayed a significant transcriptional activity and this happened only when the shunting region of the 5'-untranslated region of the 35S-sequence was omitted from the luciferase expression cassette. Gaussia-luciferase proved to be a superior quantifiable reporter gene for the analysis of constitutive promoter sequences in Chlamydomonas reinhardtii.
Center of Excellence for Fluorescent Bioanalysis, University of Regensburg, Josef-Engert-Strasse 9, Regensburg, Germany.
Ovidiu
O
Ruecker
Karina
K
Zillner
Regina
R
Groebner-Ferreira
Markus
M
Heitzer
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017796:Caulimovirus / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D033342:Copepoda / Q000201:enzymology* / Q000235:genetics*; D048028:DNA, Algal / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D017930:Genes, Reporter; D005814:Genes, Viral; D008156:Luciferases / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic
D048028:DNA, Algal; D016054:DNA, Protozoan; D008156:Luciferases