Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production
Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production
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DOI：
10.1111/pbi.12192
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发表时间：
2014-09-01
影响因子：
13.8
通讯作者：
Weeks, Donald P.
中科院分区：
文献类型：
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作者：
Bruggeman, Andrew J.;Kuehler, Daniel;Weeks, Donald P.
研究人员开发并测试了对草甘膦、羟氟醚和去氟唑酮等除草剂具有抗性的基因，作为莱茵衣藻遗传转化的优势选择标记，并作为保护商业规模藻类生产设施免受对这些广谱除草剂敏感的生物污染的潜在工具。当一个合成的草甘膦乙酰转移酶（GAT）基因与一个强衣藻启动子匹配时，转基因细胞对EPSPS抑制剂草甘膦的耐受性比前代WT细胞提高了2.7倍。一种突变的衣藻原卟啉氧化酶（原卟啉氧化酶，PPO）基因先前被证明能产生一种对PPO抑制除草剂不敏感的酶，当进行基因工程改造时，产生的转基因细胞能够比未转化的细胞耐受高达136倍的PPO抑制剂氟氧芬。对各种抗去氟拉唑生物中发现的植物烯去饱和酶（PDS）基因序列进行基因改造，使转基因细胞对去氟拉唑的耐受性比非转基因细胞高40倍。这三种抗除草剂基因在生产转基因细胞中的高效率证明了它们作为衣藻和其他真核藻类遗传转化的优势选择标记的适用性。然而，对高浓度草甘膦的要求及其对细胞生长速率的相关负面影响阻碍了对大规模生产设施使用草甘膦的考虑。相比之下，抑制细胞生长只需要低剂量的去氟唑松和羟氟芬（分别约为1.5 μ M和0.1 μ M），这表明这两种除草剂可能在大规模藻类生产设施中有效抑制对这两种除草剂敏感的生物的生长。
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 2.7x-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 136x 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 40x 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 (similar to 1.5 mu M and similar to 0.1 mu M, 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.