Enabling Genetic Transformation of Pseudo-nitzschia australis and P. multiseries

Project Overview:  
Many species within the Pseudo-nitzschia complex, including australis and P. multiseries, produce the novel neurotoxin domoic acid [DA] under a range of environmental conditions (Trainer et al., 2008). The molecular regulation and cellular localization of DA biosynthesis, which requires the interaction of amino acid and isoprene metabolism, are poorly understood (Savage et al., 2012). The ability to rapidly and precisely control gene expression in diatoms is critical for genetic manipulation and for understanding the regulation of unique metabolic pathways, such as the production of DA. The overarching goal of this proposal is to develop electroporation-based transformation methods for both P. multiseries and P. australis. This requires (1) the construction a series of vectors with selectable markers and reporter genes allowing for either constitutive and inducible expression, and (2) the development of plasmid-based, small-insert genomic libraries for P. australis to enable vector-anchored PCR isolation of regulatory domains and to assess species-specificity in transformation efficiency and expression levels. This project uses our P. australis N-source dependent transcriptome analyses (MMETSP0139-MMETSP0142) to identify genes that are either highly expressed or show inducible patterns of expression. The electroporation-based transformation approaches will facilitate future molecular physiological studies in this diatom genus and are easily transferable to other researchers as the equipment is more readily available than that needed for biolistic transformation.