Developing ribosome profiling for the marine model diatom Thalassiosira pseudonana

Pichler, Monica (2023) Developing ribosome profiling for the marine model diatom Thalassiosira pseudonana. Doctoral thesis, University of East Anglia.

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Diatoms are unicellular, eukaryotic microalgae which have evolved a vast number of regulatory mechanisms to adapt their protein synthesis in response to the changing environmental conditions they live in. Due to their key role in marine biochemical cycling and their high biotechnological potential, gene regulatory mechanism in diatoms have been extensively studied using transcriptomics and proteomics. However, regulation of protein synthesis on the translational level is largely unexplored. A ribosome profiling protocol has been developed for the model diatom Thalassiosira pseudonana which allows genome-wide analysis of translation in this globally important phytoplankton by deep sequencing of ribosome protected mRNA fragments. This method has been applied to high light stressed cells to better understand the role of translational regulation in response to changing environments. The generated dataset is the first ribosome profiling data for any marine microalgae and expands the molecular toolbox.

To study the impact of codon usage change on protein synthesis in diatoms, the codon usage of the light-harvesting complex associated Lhcx6 gene of T. pseudonana has been optimized via CRISPR/Cas9-mediated homologous recombination. Efficient gene targeting using this method has already been achieved in this species. However, this is the first time that a gene was replaced with a non-selective marker gene. Phenotyping of homozygous knock-in cell lines gives first insights into the role of codon usage in diatoms and provides preliminary data for potential future work.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Environmental Sciences
Depositing User: Chris White
Date Deposited: 14 Dec 2023 08:44
Last Modified: 14 Dec 2023 08:44


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