Thesis

Development of a genetic transformation system for the marine coccolithophore Emiliania huxleyi

The ubiquitous coccolithophore Emiliania huxleyi (E. huxley1) plays a significant role in global short-and long tenn oceanic carbon flux, warranting the recent sequencing of this organism by the United States Department of Energy Joint Genome Institute. Further progress in the study of the genomics, especially gene regulation of responses to the environment, for this keystone calcifying marine alga requires the development of a reliable method for genetic transfonnation. This report details efforts to develop a genetic transformation system for E. huxleyi. This includes the identification and characterization of a selective marker/agent system, the bialaphos resistance (BAR) gene and glufosinate ammonium; the development of DNA constructs containing the BAR gene driven by an endogenous promoter; testing of DNA delivery tools (primarily microparticle bombardment); post treatment selective culture for enrichment of potential transformants; and verification of transformation using real-time PCR and Western blots. Although the evidence of transient expression of foreign genes in the treated Emiliania huxleyi strain 1516 cells. cells was promising, and our efforts help pave the way for further development, more work is needed to secure a reliable genetic transfonnation system for E. huxleyi. Keywords: Haptophyte algae, coccolithophore, Emiliania huxleyi, genetic transformation, electroporation, microparticle bombardment, bialaphos resistance, BAR gene, glufosinate ammonium.

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