Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast
Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast
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DOI：
10.1046/j.1365-313x.2003.01965.x
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发表时间：
2004-02-01
期刊：
影响因子：
7.2
通讯作者：
Schultz, J
中科院分区：
文献类型：
--
作者：
Mayfield, SP;Schultz, J
荧光素酶报告基因已成功地用于多种生物体中以检测活细胞中的基因表达，但尚未成功地开发用于叶绿体中。绿色荧光蛋白（gfp）已被用作叶绿体基因表达的报告基因，但由于高的自发荧光，需要非常高水平的GFP积累以用于体内可视化。我们已经开发了一个叶绿体荧光素酶报告基因，通过合成两个亚基的细菌荧光素酶（lux）AB，作为一个单一的融合蛋白在莱茵衣藻叶绿体密码子偏好。我们在C.在ATP酶α亚基（atpA）或psbA启动子和5'非翻译区（UTR）和rubisco大亚基（rbcL）3' UTR的控制下，将ATP酶α亚基（atpA）或psbA启动子和5'非翻译区（UTR）连接到莱茵衣藻叶绿体中。我们表明，luxCt是一个敏感的报告叶绿体基因表达，荧光素酶活性可以在体内使用电荷耦合器件（CCD）相机或在体外使用光度计测量。我们进一步证明，luxCt蛋白质的积累，通过Western印迹分析，是成比例的发光，在体内和体外确定，luxCt是能够报告的变化，叶绿体基因表达在黑暗中，以光的转变。这些数据证明了luxCt基因作为活细胞中叶绿体基因表达的通用和敏感的报告基因的效用。
Luciferase reporter genes have been successfully used in a variety of organisms to examine gene expression in living cells, but are yet to be successfully developed for use in chloroplast. Green fluorescent protein (gfp) has been used as a reporter of chloroplast gene expression, but because of high auto-fluorescence, very high levels of GFP accumulation are required for visualization in vivo. We have developed a luciferase reporter for chloroplast by synthesizing the two-subunit bacterial luciferase (lux)AB, as a single fusion protein in Chlamydomonas reinhardtii chloroplast codon bias. We expressed a chloroplast luciferase gene, luxCt, in C. reinhardtii chloroplasts under the control of the ATPase alpha subunit (atpA) or psbA promoter and 5' untranslated regions (UTRs) and the rubisco large subunit (rbcL) 3' UTR. We show that luxCt is a sensitive reporter of chloroplast gene expression, and that luciferase activity can be measured in vivo using a charge coupled device (CCD) camera or in vitro using a luminometer. We further demonstrate that luxCt protein accumulation, as measured by Western blot analysis, is proportional to luminescence, as determined both in vivo and in vitro, and that luxCt is capable of reporting changes in chloroplast gene expression during a dark to light shift. These data demonstrate the utility of the luxCt gene as a versatile and sensitive reporter of chloroplast gene expression in living cells.