Phytoplankton-group specific quantitative polymerase chain reaction assays for RuBisCO mRNA transcripts in seawater.
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.
John
David E
DE
College of Marine Science, University of South Florida, Saint Petersburg, FL 33701, USA. djohn@marine.usf.edu
Patterson
Stacey S
SS
Paul
John H
JH
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
D015342:DNA Probes; D012333:RNA, Messenger; D012273:Ribulose-Bisphosphate Carboxylase
Appl Environ Microbiol. 2006 Jan;72(1):723-32
16391112
Appl Environ Microbiol. 2004 Feb;70(2):1199-206
14766606
Appl Environ Microbiol. 2004 Sep;70(9):5459-68
15345433
Appl Environ Microbiol. 2004 Aug;70(8):4727-32
15294808
Appl Environ Microbiol. 2000 Jun;66(6):2349-57
10831410
Appl Environ Microbiol. 2005 Mar;71(3):1659-61
15746374
FEMS Microbiol Ecol. 2005 Mar 1;52(1):79-92
16329895
Plant Cell Environ. 2006 Nov;29(11):2043-54
17081240
Appl Environ Microbiol. 2002 Aug;68(8):3771-9
12147471
Science. 2004 May 14;304(5673):1005-8
15143279
Brief Bioinform. 2004 Jun;5(2):150-63
15260895
Environ Microbiol. 2006 Mar;8(3):441-54
16478451
Appl Environ Microbiol. 2006 Apr;72(4):2496-506
16597949