Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene.
For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii . This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii .
Fuhrmann
Markus
M
Institut für Biochemie I, Universität Regensburg, 93040 Regensburg, Germany. markus.fuhrmann@vkl.uni-regensburg.de
Hausherr
Amparo
A
Ferbitz
Lars
L
Schödl
Thomas
T
Heitzer
Markus
M
Hegemann
Peter
P
D020418:Algal Proteins / Q000235:genetics; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007093:Imidazoles / Q000378:metabolism; D008156:Luciferases / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011719:Pyrazines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection
D020418:Algal Proteins; D007093:Imidazoles; D009687:Nuclear Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D011719:Pyrazines; D011993:Recombinant Fusion Proteins; C017144:coelenterazine; D008156:Luciferases; D012273:Ribulose-Bisphosphate Carboxylase