Transformation of Diplonema papillatum, the type species of the highly diverse and abundant marine microeukaryotes Diplonemida (Euglenozoa)

0498712 - BC 2019 RIV GB eng J - Journal Article
Kaur, Binnypreet - Valach, M. - Peña-Diaz, Priscila - Moreira, S. - Keeling, P. J. - Burger, G. - Lukeš, Julius - Faktorová, Drahomíra
Transformation of Diplonema papillatum, the type species of the highly diverse and abundant marine microeukaryotes Diplonemida (Euglenozoa).
Environmental Microbiology. Roč. 20, č. 3 (2018), s. 1030-1040. ISSN 1462-2912. E-ISSN 1462-2920
R&D Projects: GA ČR GA15-21974S; GA ČR(CZ) GA16-18699S; GA MŠMT LL1601
Institutional support: RVO:60077344
Keywords : TRYPANOSOMA-BRUCEI * GENE-EXPRESSION * PROTEINS * DNA * KINETOPLASTIDA * RECOMBINATION * TRANSFECTION * EUKARYOTES
OECD category: Genetics and heredity (medical genetics to be 3)
Impact factor: 5.147, year: 2018

Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd 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.
Permanent Link: http://hdl.handle.net/11104/0290994