Shao Ning, Bock Ralph
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
Curr Genet. 2008 Jun;53(6):381-8. doi: 10.1007/s00294-008-0189-7. Epub 2008 Apr 12.
The unicellular green alga Chlamydomonas reinhardtii has emerged as a superb model species in plant biology. Although the alga is easily transformable, the low efficiency of transgene expression from the Chlamydomonas nuclear genome has severely hampered functional genomics research. For example, poor transgene expression is held responsible for the lack of sensitive reporter genes to monitor gene expression in vivo, analyze subcellular protein localization or study protein-protein interactions. Here, we have tested the luciferase from the marine copepod Gaussia princeps (G-Luc) for its suitability as a sensitive bioluminescent reporter of gene expression in Chlamydomonas. We show that a Gaussia luciferase gene variant, engineered to match the codon usage in the Chlamydomonas nuclear genome, serves as a highly sensitive reporter of gene expression from both constitutive and inducible algal promoters. Its bioluminescence signal intensity greatly surpasses previously developed reporters for Chlamydomonas nuclear gene expression and reaches values high enough for utilizing the reporter as a tool to monitor responses to environmental stresses in vivo and to conduct high-throughput screenings for signaling mutants in Chlamydomonas.
单细胞绿藻莱茵衣藻已成为植物生物学中一个出色的模式物种。尽管这种藻类易于转化,但来自莱茵衣藻核基因组的转基因表达效率低下严重阻碍了功能基因组学研究。例如,转基因表达不佳导致缺乏灵敏的报告基因来监测体内基因表达、分析亚细胞蛋白质定位或研究蛋白质-蛋白质相互作用。在此,我们测试了来自海洋桡足类动物王子高斯藻(G-Luc)的荧光素酶作为莱茵衣藻中基因表达的灵敏生物发光报告基因的适用性。我们表明,经过工程改造以匹配莱茵衣藻核基因组密码子使用情况的高斯荧光素酶基因变体,可作为组成型和诱导型藻类启动子基因表达的高度灵敏报告基因。其生物发光信号强度大大超过了先前开发的用于莱茵衣藻核基因表达的报告基因,达到了足以将该报告基因用作监测体内对环境胁迫的反应以及对莱茵衣藻信号突变体进行高通量筛选工具的值。