title: Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production.
abstract: Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial-scale algal production facilities against contamination by organisms sensitive to these broad-spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×-fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO-inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene-based gene sequences found in various norflurazon-resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large-scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 μm and ~0.1 μm, respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large-scale algal production facilities in suppressing growth of organisms sensitive to these herbicides.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Brueggeman
authors.forename: Daniel
authors.initials: D
authors.lastname: Kuehler
authors.forename: Donald P
authors.initials: DP
authors.lastname: Weeks
mesh_terms: D000123:Acetyltransferases / Q000235:genetics*; D000595:Amino Acid Sequence; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000235:genetics*; D018556:Crops, Agricultural / Q000187:drug effects / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D055768:Halogenated Diphenyl Ethers / Q000494:pharmacology; D053209:Herbicide Resistance / Q000235:genetics*; D006540:Herbicides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000187:drug effects; D050768:Protoporphyrinogen Oxidase / Q000235:genetics*; D011724:Pyridazines / Q000494:pharmacology; D016415:Sequence Alignment; D014170:Transformation, Genetic; D000097797:Glyphosate
chemical_list: D000123:Acetyltransferases; D005998:Glycine; D055768:Halogenated Diphenyl Ethers; D006540:Herbicides; D010088:Oxidoreductases; D010940:Plant Proteins; D050768:Protoporphyrinogen Oxidase; D011724:Pyridazines; D000097797:Glyphosate; C016027:oxyfluorofen; C022398:phytoene dehydrogenase; C486861:glyphosate N-acetyltransferase; C008677:norflurazone
keywords: Algae; Chlamydomonas reinhardtii; glyphosate; herbicide resistance; norflurazon; oxyfluorfen