Real-time PCR quantification of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) mRNA in diatoms and pelagophytes.
Transcriptional activity is often used as a surrogate for gene expression in environmental microbial communities. We developed a real-time PCR assay in which the ABI-Prism (PE Applied Biosystems) detection system is used for quantification of large-subunit ribulose-1,5-bisphosphate caboxylase/oxygenase (rbcL) mRNA in diatoms and pelagophytes both in cultures and from natural phytoplankton communities. Plasmid DNA containing rbcL inserts, as well as in vitro transcribed mRNA of the plasmids, was used to generate standard curves with a dynamic range of more than 6 orders of magnitude with high accuracy and precision (R(2) = 0.998). Expression levels in a cultured diatom (Phaeodactylum tricornutum) were quantified through one light-dark cycle by using traditional 35S-labeled oligonucleotide hybridization and real-time PCR. The mRNA levels detected by the two techniques were similar and correlated well (R(2) = 0.95; slope = 1.2). The quantities obtained by hybridization were slightly, yet significantly, larger (t = 5.29; P = 0.0011) than the quantities obtained by real-time PCR. This was most likely because partially degraded transcripts were not detected by real-time PCR. rbcL mRNA detection by real-time PCR was 3 orders of magnitude more sensitive than rbcL mRNA detection by hybridization. Diatom and pelagophyte rbcL mRNAs were also quantified in a profile from an oligotrophic site in the Gulf of Mexico. We detected the smallest amount of diatom rbcL expression in the surface water and maximum expression at a depth that coincided with the depth of the subsurface chlorophyll maximum. These results indicate that real-time PCR may be utilized for quantification of microbial gene expression in the environment.
Wawrik
B
B
College of Marine Science, University of South Florida, St. Petersburg, Florida 33701, USA.
Paul
J H
JH
Tabita
F R
FR
D000818:Animals; D003470:Culture Media; D015342:DNA Probes; D017377:Diatoms / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D056890:Eukaryota / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005434:Flow Cytometry; D009693:Nucleic Acid Hybridization / Q000379:methods; D010839:Phytoplankton / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism*; D012623:Seawater / Q000382:microbiology; D013045:Species Specificity; D019914:Taq Polymerase / Q000378:metabolism
D003470:Culture Media; D015342:DNA Probes; D012333:RNA, Messenger; D019914:Taq Polymerase; D012273:Ribulose-Bisphosphate Carboxylase