Graduation Year

2007

Document Type

Thesis

Degree

M.S.

Degree Granting Department

Marine Science

Major Professor

John H. Paul, III, Ph.D.

Keywords

Phytoplankton, CBB Pathway, Mississippi River Plume, RbcL, Pelagophyte, Synechococcus

Abstract

The Calvin-Benson-Bassham (CBB) pathway is the primary pathway for the entry of inorganic carbon in the biosphere. Autotrophic organisms use this cycle to ultimately convert CO2 into carbohydrates using a key enzyme known as ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene that encodes for the large subunit of RubisCO is rbcL and detection of its expression can be used to determine the autotrophic organisms present in the environment. Recently, microarrays have been used to study functional gene expression from environmental samples such as those obtained from sediments and soil. The purpose of this thesis is to combine microarray technology and rbcL expression analysis to investigate phytoplankton populations in the Mississippi River Plume (MRP). Initially, a macroarray was constructed to determine its capabilities of quantifying gene expression in MRP.

PCR amplicons were spotted onto a nylon membrane and labeled transcript RNA was hybridized to each array. Due to the large amount of cross hybridization that was observed, a microarray was used. Microarray analysis revealed large amounts of Synechococcus, pelagophyte and prymnesiophyte expression in the surface waters. Furthermore, there was no chlorophte or Prochlorococcus expression observed in the surface waters. Subsurface microarray data showed high levels of pelagophytes and other Form ID organisms. A significant chlorophyte signal was also observed in the subsurface. This study provides a third level of specificity at which phylogenetic diversity has been sampled in the MRP. Although a limited number of samples were analyzed by microarrays, this technology shows promise and this study was viewed as a pilot for their application.

The rbcL probes designed were based upon published sequences from 2003 and we now have a much greater understanding of the diversity of rbcL-containing phytoplanktonic phylotypes. Future studies should employ this knowledge for judicious probe selection.

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