Abstract
Diatoms are ubiquitous primary producers in marine ecosystems and freshwater habitats. Due to their complex evolutionary history, much remains unknown about the specific gene functions in diatoms that underlie their broad ecological success. In this study, we have genetically transformed the centric diatom Skeletonema marinoi, a dominant phytoplankton species in temperate coastal regions. Transformation of S. marinoi is the first for a true chain-forming diatom, with the random genomic integration via nonhomologous recombination of a linear DNA construct expressing the resistance gene to the antibiotic zeocin. A set of molecular tools were developed for reliably identifying the genomic insertion site within each transformant, many of which disrupt recognizable genes and constitute null or knock-down mutations. We now propose S. marinoi as a new genetic model for marine diatoms, representing true chain-forming species that play a central role in global photosynthetic carbon sequestration and the biogeochemical cycling of silicates and various nutrients, as well as having potential biotechnological applications.
Highlights
Phytoplankton are the foundation of the marine food web that sustains most oceanic life
Genetic transformation of diatoms has been shown for several pennate species using mainly microparticle bombardment[18,19,20,21,22,23,24] and electroporation[25,26] to deliver relatively long, linearized plasmid DNA that randomly insert into the nuclear genome by nonhomologous recombination
Fewer examples exist for transformation of centric diatoms and none for true chain-forming species such as S. marinoi
Summary
Phytoplankton are the foundation of the marine food web that sustains most oceanic life. The nuclear genomes of several different diatoms have been sequenced[8,9,10,11,12], including the pennate Phaeodactylum tricornutum and centric Thalassiosira pseudonana These sequences have revealed a large proportion of hypothetical proteins of unknown function The most successful genetic strategy for elucidating the specific function of these numerous hypothetical proteins in many different organisms has been mutagenesis using either forward or reverse genetics In this regard, large collections of random insertional mutants suitable for both single gene studies and large-scale phenotyping have been of particular value as exemplified by the vast volume of knowledge gained over many years from the T-DNA mutant collections for the vascular plant Arabidopsis thaliana[15]. As proof-of-concept of the suitability of S. marinoi for future mutagenesis studies, the insertion site in selected transformants were mapped, in which most occurred within recognizable gene structures. Ca. one-third were shown to be homozygous for the mutation, with the others being heterozygous
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