Abstract
The unicellular red alga Cyanidioschyzon merolae possesses a simple cellular architecture that consists of one mitochondrion, one chloroplast, one peroxisome, one Golgi apparatus, and several lysosomes. The nuclear genome content is also simple, with very little genetic redundancy (16.5 Mbp, 4,775 genes). In addition, molecular genetic tools such as gene targeting and inducible gene expression systems have been recently developed. These cytological features and genetic tractability have facilitated various omics analyses. However, only a single transformation selection marker URA has been made available and thus the application of genetic modification has been limited. Here, we report the development of a nuclear targeting method by using chloramphenicol and the chloramphenicol acetyltransferase (CAT) gene. In addition, we found that at least 200-bp homologous arms are required and 500-bp arms are sufficient for a targeted single-copy insertion of the CAT selection marker into the nuclear genome. By means of a combination of the URA and CAT transformation systems, we succeeded in producing a C. merolae strain that expresses HA-cyclin 1 and FLAG-CDKA from the chromosomal CYC1 and CDKA loci, respectively. These methods of multiple nuclear targeting will facilitate genetic manipulation of C. merolae.
Highlights
Eukaryotic algae synthesize organic compounds such as carbohydrates and amino acids from inorganic materials by using light energy, and introduce organic products into aquatic ecosystems
In order to validate the application of the chloramphenicol acetyltransferase (CAT) gene as a selection marker for the nuclear transformation of C. merolae by homologous recombination, we constructed a CAT selection marker cassette consisting of the APCC promoter, a chloroplasttransit peptide, the CAT orf and the β-tubulin 3 utr
In order to integrate the CAT selection marker cassette into the convergent intergenic region of CMD184C and CMD185C as a neutral chromosomal locus by homologous recombination, the cassette was flanked with ∼1.5-kb chromosomal sequences (Figure 1A)
Summary
Eukaryotic algae synthesize organic compounds such as carbohydrates and amino acids from inorganic materials by using light energy, and introduce organic products into aquatic ecosystems. Unicellular algae are potentially useful for other basic sciences in several different disciplines and applied fields for the following reasons: (1) cultures of unicellular algae often provide a homogeneous population of a single cell type, in contrast to land plants in which complicated cell and tissue differentiation systems have evolved, (2) in algal culture, each cell is exposed to a relatively homogeneous environment (light, inorganic nutrient concentrations, etc.), while each land plant cell is exposed to different environments, in the same individual, (3) unicellular eukaryotic algae often exhibit relatively simple cellular and genomic architectures, (4) the generation time required for unicellular algae is much shorter than for land plants, (5) unicellular algae are widespread in tree of life owing to secondary endosymbiotic events in which previously non-photosynthetic eukaryotes acquired chloroplasts through endosymbiotic association with eukaryotic algal endosymbionts (Reyes-Prieto et al, 2007), (6) unicellular algae are being developed that are able to produce nutraceuticals and alternative energy (Radakovits et al, 2010) These characteristics of unicellular algae potentially offer ideal experimental platforms, but experimental tools, especially those for genetic modification of eukaryotic algae, have remained extremely limited. Instability of transgene expression because of gene silencing is still a serious problem (Tracey and Sohail, 2016)
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