Diplonema papillatum is the type species of diplonemids, which are among the most abundant and diverse heterotrophic microeukaryotes in the world's oceans. Diplonemids are also known for a unique form of post-transcriptional processing in mitochondria. However, the lack of reverse genetics methodologies in these protists has hampered elucidation of their cellular and molecular biology. Here we report a protocol for D. papillatum transformation. We have identified several antibiotics to which D. papillatum is sensitive and thus are suitable selectable markers, and focus in particular on puromycin. Constructs were designed encoding antibiotic resistance markers, fluorescent tags, and additional genomic sequences from D. papillatum to facilitate vector integration into chromosomes. We established conditions for effective electroporation, and demonstrate that electroporated constructs can be stably integrated in the D. papillatum nuclear genome. In D. papillatum transformants, the heterologous puromycin resistance gene is transcribed into mRNA and translated into protein, as determined by Southern hybridization, reverse transcription, and Western blot analyses. This is the first documented case of transformation in a euglenozoan protist outside the well-studied kinetoplastids, making D. papillatum a genetically tractable organism and potentially a model system for marine microeukaryotes.

• MeSH
• Euglenozoa genetika   fyziologie   MeSH
• Eukaryota genetika   MeSH
• fylogeneze MeSH
• léková rezistence MeSH
• messenger RNA genetika   metabolismus   MeSH
• mitochondrie MeSH
• puromycin farmakologie   MeSH
• regulace genové exprese MeSH
• transformace genetická * MeSH
• vodní organismy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH