Molecular Investigation of Candidate Genes for the Biosynthetic Pathway for Dimethylsulfoniopropionate (DMSP) in the Diatom Thalassiosira pseudonana
Dimethylsulfoniopropionate (DMSP) is a sulfuric zwitterionic compound produced primarily by marine phytoplankton. DMSP is the main biogenic precursor for the climatically active gas dimethyl sulfide (DMS), and is involved in the assimilation of sulfur in marine food webs. In marine algae, DMSP is a multifunctional molecule involved in stress adaptations, as it may function as an osmolyte, cryoprotectant, antioxidant and as a chemical defense molecule against grazing zooplankton. Five candidate genes have been proposed to be involved in the poorly understood biosynthesis of DMSP in marine algae, featuring the four enzymatic reactions transamination, reduction, S-methylation and oxidative decarboxylation. In this thesis, the candidate genes DiDECARB, SAMmt, REDOX and AT were investigated for their role in the DMSP biosynthetic pathway in the diatom Thalassiosira pseudonana. As studies have shown that DMSP accumulates in marine algae under nutrient limitations, the gene expression of these candidate genes was examined using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), and the hypothesized DMSP accumulation was measured by gas chromatography-mass spectrometry (GC-MS), when diatom cultures were grown under the limitations of the nutrient silicate and nitrate. The candidate genes were also similarly investigated under complete silicate starvation. Attempts to optimize total RNA isolation of T. pseudonana for RT-qPCR were made. The results indicated that the QIAGEN Protocol B was the most suitable total RNA purification protocol tested, and that the Durapore® DVPP membrane filter (Merck Millipore) for cell harvesting by vacuum filtration resulted in the highest RNA yield.
The results obtained in this thesis, indicated that DMSP failed to accumulate under nutrient limitations of silicate and nitrate, but was significantly accumulated during complete silicate starvation. The candidate gene REDOX was found to be significantly up-regulated after 5 days of nitrate limitation, and the DiDECARB gene was significantly up-regulated after 72 hours of complete silicate starvation, although not strongly statistically supported. The results obtained in this thesis were not able to verify that the candidate genes are involved in the biosynthesis of DMSP. Although a link between the proposed candidate genes and the biosynthesis of DMSP cannot be ruled out, further research is needed to reveal the role of the candidate genes and/or find new candidate genes for the biosynthetic pathway for DMSP in marine algae.
Place, publisher, year, edition, pages
Institutt for biologi , 2013. , 132 p.
IdentifiersURN: urn:nbn:no:ntnu:diva-21605Local ID: ntnudaim:10223OAI: oai:DiVA.org:ntnu-21605DiVA: diva2:640172
Bones, Atle M., ProfessorBrembu, Tore