Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast.
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.
Mayfield
Stephen P
SP
Department of Cell Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA. mayfield@scripps.edu
Schultz
Jason
J
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics*; D003062:Codon; D017930:Genes, Reporter; D008027:Light; D008156:Luciferases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D003062:Codon; D012333:RNA, Messenger; D008156:Luciferases