Transformation of Diplonema papillatum, the type species of the highly diverse and abundant marine microeukaryotes Diplonemida (Euglenozoa).
Environ Microbiol
; 20(3): 1030-1040, 2018 03.
Article
em En
| MEDLINE
| ID: mdl-29318727
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Transformação Genética
/
Euglenozoários
Idioma:
En
Revista:
Environ Microbiol
Ano de publicação:
2018
Tipo de documento:
Article