Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast.
Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5'- and 3'-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.
Franklin
Scott
S
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. sefrankl@scripps.edu
Ngo
Binh
B
Efuet
Ekem
E
Mayfield
Stephen P
SP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism; D018742:DNA, Chloroplast / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D030781:Organisms, Genetically Modified; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D018742:DNA, Chloroplast; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins