Development of a luciferase reporter gene, luxCt , for Chlamydomonas reinhardtii chloroplast

Summary 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.