title: Phytoplankton-group specific quantitative polymerase chain reaction assays for RuBisCO mRNA transcripts in seawater.
abstract: The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) has been shown to be a useful target for molecular assays that quantify form- or clade-specific RNA transcript concentrations as a proxy for the carbon fixation activity of marine phytoplankton. To improve the phylogenetic specificity and sensitivity of RNA probe hybridization methods, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay has been reported for diatom and pelagophyte rbcL RNA. Here we detail enhancements made to this PCR method and development of additional assays to specifically quantify rbcL expression from haptophytes, Synechococcus and high-light Prochlorococcus. In vitro RNA transcripts were tested to demonstrate specificity and quantitative accuracy. Application of these methods on seawater samples from two depth profiles in the northern Gulf of Mexico showed a fair degree of agreement between PCR and hybridization results, with results for the chromophytic or form ID rbcL-containing organisms having better agreement between the two methods. Diatoms and other heterokonts were shown to be the primary carbon fixers at these locations by PCR, in agreement with greater form ID rbcL RNA measured by hybridization.
authors: College of Marine Science, University of South Florida, Saint Petersburg, FL 33701, USA. djohn@marine.usf.edu
authors: David E
authors: DE
authors: John
authors: Stacey S
authors: SS
authors: Patterson
authors: John H
authors: JH
authors: Paul
mesh_terms: D015342:DNA Probes / Q000737:chemistry; D017377:Diatoms; D004777:Environment; D015964:Gene Expression Regulation, Bacterial; D010802:Phylogeny; D010839:Phytoplankton / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D048929:Prochlorococcus / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis / Q000235:genetics*; D012623:Seawater / Q000032:analysis*; D012680:Sensitivity and Specificity; D046940:Synechococcus / Q000235:genetics
chemical_list: D015342:DNA Probes; D012333:RNA, Messenger; D012273:Ribulose-Bisphosphate Carboxylase
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