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ABSTRACT
In the past, various species of the unicellular algal genus Micrasterias (Zygnematophyceae) have been used for research in the fields of cell biology and physiology. Relatively large cell size, highly differentiated cell shape and a remarkable evolutionary position make Micrasterias interesting, especially for cytomorphogenetic studies. However, within this genus a model organism enabling molecular research has not yet been established. Micrasterias radians var. evoluta (W.B.Turner) Krieger allows easy culturing under axenic conditions and performs its whole life cycle in vitro thus fulfilling two important prerequisites for a model organism. In this work resources allowing transient expression of transgenes were developed. First, axenic lines were obtained by the treatment of zygospores with a cocktail of antibiotics followed by germination and regeneration. In order to allow transgene expression we isolated the 5′ -flanking region of the M. radians var. evoluta Actin1 gene (MrACT1) and fused it to the green fluorescent protein (GFP). A higher promoter activity compared with various heterologous promoters regarding GFP expression was observed. Transient transgene expression was achieved by polyethylene glycol (PEG)-mediated protoplast transformation, yielding a rate of 3.9% transformed cells per surviving protoplasts. Transgene expression was also achieved by particle bombardment of vegetative cells. Employing the established protocol, a Lifeact-GFP fusion protein for labelling F-actin was expressed, allowing visualization of the actin cytoskeleton in M. radians var. evoluta.
Acknowledgements
The authors thank H. Sekimoto (Japan Women’s University) for kindly sharing the constructs pSA405A and H. Buschmann (Osnabrück University) for gifting plasmid pMD as well as scientific exchange about the bombardment technique. Technical assistance by V. Schwekendiek and S. Körner is gratefully acknowledged.
Disclosure statement
No potential conflict of interest was reported by the authors.
Supplementary information
The following supplementary material is accessible via the Supplementary Content tab on the article’s online page at https://doi.org/10.1080/09670262.2020.1768298
Supplementary table 1: The composition of C-medium used for Micrasterias radians var. evoluta cultivation.
Supplementary table 2: Primers used for amplification of partial coding sequence and 5′ flanking region of Actin1 gene from Micrasterias radians var. evoluta.
Supplementary fig. 1: Alignment of partial of Micrasterias radians var. evoluta Actin1 genomic DNA (gDNA) sequence and nucleotide sequence obtained by the ligation based 5′ RACE-PCR (5′ RACE).
Supplementary fig. 2: Alignment of the partial coding sequences from Actin genes of various organisms and partial MrACT1 gDNA sequence.
Author contributions
H. Zhou: design of the project, genetic transformation of M. radians var. evoluta protoplasts and vegetative cells, writing of manuscript, reviewing and developing the manuscript for submission. A. Wilkens: experiments for purification of the axenic culture, isolation and characterization of the endogenous promoter, genetic transformation of M. radians var. evoluta protoplasts and vegetative cells, reviewing and developing the manuscript for submission. D. Hanelt: reviewing and developing the manuscript for submission. K. von Schwartzenberg: design of the project, reviewing and developing the manuscript for submission.
Supplemental Material
The following supplementary material is accessible via the Supplementary Content tab on the article’s online page at https://doi.org/10.1080/09670262.2020.1768298.