Stable nuclear transformation methods for Euglena gracilis and its application to a related Euglenida

Euglenida is a taxonomic group of single-celled flagellates that have a variety of nutrient uptake strategies, making this an excellent model for studying the evolutionary acquisition of secondary chloroplasts. Among Euglenida, Euglena gracilis is the most extensively studied at the biochemical and molecular levels. However, the lack of a widely adopted method of nuclear genome transformation has hindered genetic studies in this organism. Herein, we present a novel nuclear transformation electroporation method that utilizes the 5′ adjacent region sequences of endogenous genes in E. gracilis. We used a NanoLuc reporter to evaluate promoter activity to identify four endogenous promoter candidates with superior luciferase transcriptional activity compared with the activity of the commonly used CaMV 35S promoter in E. gracilis nuclear transformation methods. We used G418 selection to obtain stable E. gracilis transformants harboring the transgene in their genomic DNA. Furthermore, we extended the applicability of our method to Rapaza viridis, a kleptoplastic species in Euglenida. Introduction of the DNA constructs developed for E. gracilis into R. viridis via electroporation generated stable G418-resistant strains exhibiting robust luciferase activity. The introduced promoter sequences functioned effectively, and accurate 5′ end modifications of mRNA were observed in both E. gracilis and R. viridis transformants. These findings demonstrate the broad applicability of our nuclear transformation method across multiple Euglenida species, facilitating molecular biology and genetic diversity studies in this taxonomic group.