Assessing Primary Production in Diatoms Using Genetic RealTime PCR and Biogeochemical 14C Fixation T

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Assessing Primary Production in Diatoms Using
Genetic (Real-Time PCR) and Biogeochemical (14C
Fixation) TechniquesA. Lorenzi, M. Takabayashi,
and F.P. Wilkerson
Romberg Tiburon Center, SFSU, Tiburon, CA
Abstract Real time reverse transcription PCR, a
new technology to the marine field, is capable of
detecting low quantities of nucleic acid. This
technique was applied to studies of phytoplankton
communities in Central San Francisco Bay (CFSB).
Wawrik et al (2002) have successfully measured
mRNA transcript expression of RubisCO (rbcL) in
chromophytes in the marine environment. Similar
application of RT-PCR in CSFB will yield a
database of transcriptional regulation
measurements by the diatom community (i.e. cells
with diameters gt5µm). In addition, carbon-14
uptake data have been collected to examine the
biogeochemical side of primary productivity. By
taking both molecular and community-level
approaches, we can quantitatively link primary
productivity and diatoms to illustrate their
ecological contribution to estuarine production.
Biogeochemical Approach
Genetic Approach
Standard Curve
A pair of degenerate primers and a Taqman probe
assay have been developed for rbcL (the gene that
encodes the large subunit in the major enzyme
responsible for carbon fixation, RubisCO) in
chromophytes and pelagophytes. Our goal is to use
absolute RT-PCR to create a database of
measurements of the transcriptional activity of
RubisCO to compare directly with 14C primary
productivity measurements.
Chl-a
Form ID rbcL chromophytes and pelagophytes Forwar
d Primer 5-GATGATGARAAYATTAACTC-3 Reverse
Primer 5-ATTTGDCCACAGTGDATACCA-3 Taqman Probe
5-TGCGTTGGAGAGAGAICGTTTCTTA-3 (Wawrik et al,
2002)

Standard curve created via cloning of rbcL
transcript into E. coli and creating serially
diluted cDNA for RT-PCR is repeatable.
RT-PCR Reaction The Mechanics
Real time PCR utilizes a standard PCR cycle but
incorporates a fluorescent dye reaction to
quantify a mRNA transcript number or template. A
Taqman probe assay (designed here for diatoms)
hybridizes to rbcL mRNA transcripts and as Taq
extends, it cleaves a reporter dye (FAM) which
disassociates away from a quencher dye (TAMRA)
eliciting a measurable fluorescent signal.
CSFB Samples
Conclusions
?There are few direct 14C measurements for
Central San Francisco Bay most published primary
productivity values are derived from a simple
irradiance based model (Cole and Cloern, 1987).
These direct measurements show Central SFB to
have low levels of primary productivity compared
to other estuaries. ?Real-time RT-PCR for rbcL
for diatoms can be applied to Central San
Francisco Bay samples. ?RT-PCR is capable of
detecting mRNA molecules from a distinct class of
phytoplankton but its sensitivity may be
problematic when working with replicates from the
field due to reaction inefficiencies in
situations with low concentrations of the target
sequence. ?Investigating transcriptional
regulation of diatoms in Central San Francisco
Bay will aid further understanding of the factors
regulating primary production.
Results ?Samples collected from Central San
Francisco Bay in October show high mRNA
transcript numbers (low Ct values) indicating
high expression of rbcL in diatoms. ?Triplicates
of 10/14/04 sample shows variability of Ct value
which is likely due to reaction inefficiencies
compared to tight Ct data of 10/11/04. ?No
template control indicates little to no
contamination by genomic DNA. ?The failed
reaction occurred in a sample that contained no
diatoms (as observed with microscopy). ?Samples
(10/11/04, 10/14/04, and 10/15/04) exhibit a
decrease in mRNA transcript number (Ct values
increase) indicating either a decrease in
transcription or decrease in cell abundance.
However, there are inconsistencies between
runs. We have been unsuccessful in simultaneously
measuring amplification in samples and the
standard curve. Note Cycle Threshold (Ct) is
an arbitrary line drawn to best fit to data.
Applied Biosystems
Absolute vs. Relative Method of RT-PCR
Absolute Requires standard curve to be made of
target sequence for comparison against field
samples. Relative Uses an endogenous
housekeeping gene (one that is transcribed at the
same rate under all environmental conditions) to
measure against change of target
transcript. Rationale for this project Absolute
quantification was used due to lack of an
endogenous control gene probe from chromophytes.
References Cole, B. E. and Cloern, J. E. 1987. An
empirical model for estimating phytoplankton
productivity in estuaries. Mar. Ecol. Prog. Ser.
36299-305. Wawrik, B., Paul, J.H., Tabita, F.R.
2002. Real-time PCR quantification of rbcL
(Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase)
mRNA in Diatoms and Pelagophytes. Appl. Environ.
Microbiol. 683771-3779.