Abstract
The unicellular red alga Cyanidioschyzon merolae is a model organism for studying the basic biology of photosynthetic organisms. The C. merolae cell is composed of an extremely simple set of organelles. The genome is completely sequenced. Gene targeting and a heat-shock inducible gene expression system has been recently established. However, a conditional gene knockdown system has not been established, which is required for the examination of function of genes that are essential to cell viability and primary mutant defects. In the current study, we first evaluated the expression of a transgene from two chromosomal neutral loci located in the intergenic region between CMD184C and CMD185C, and a region upstream of the URA5.3 gene. There was no significant difference in expression between them and this result suggests that both may be used as neutral loci. We then designed an inducible and repressible gene expression by using promoters of nitrate-assimilation genes. The expression of nitrate-assimilation genes such as NR (nitrate reductase), NIR (nitrite reductase), and NRT (the nitrate/nitrite transporter) are reversibly regulated by their dependence on nitrogen sources. We constructed stable strains in which a cassette containing the NR, NIR, or NRT promoter and sfGFP gene was inserted in a region upstream of URA5.3 and examined the efficacy of the promoters. The NR, NIR, and NRT promoters were constitutively activated in the nitrate medium, whereas their activities were extremely low in presence of ammonium. The activation of each promoter was immediately inhibited within a period of 1 h by the addition of ammonium. Thus, a conditional knockdown system in C. merolae was successfully established. The activity varies among the promoters, and each is selectable according to the expression level of a target gene estimated by RNA-sequencing. This method is applicable to defects in genes of interest in photosynthetic organism.
Highlights
In studies on photosynthetic eukaryotes, molecular genetic techniques have been developed and used extensively for certain land plants and the unicellular green alga Chlamydomonas reinhardtii widely used as model systems
Estimation of an Ability to Express Transgene of Two Different Neutral Loci by Comparing strain in which the APCC promotersuperfolder GFP (sfGFP) Expression In order to evaluate the activities of promoters of nitrateassimilation genes, we planned to express reporter protein [green fluorescent protein (GFP)] under the control of respective promoters from a C. merolae neutral chromosomal locus
We previously prepared a strain in which the APCC promotersuperfolder GFP and URA5.3 genes are integrated into the intergenic region between CMD184C and CMD185C of the C. merolae uracil-auxotrophic strain M4, which possesses a point mutation in the chromosomal URA5.3 locus (Sumiya et al, 2014; termed D-APCCp in this study; Figure 1A)
Summary
In studies on photosynthetic eukaryotes, molecular genetic techniques have been developed and used extensively for certain land plants and the unicellular green alga Chlamydomonas reinhardtii widely used as model systems. For the investigation of phenomena that are generally shared by algae and land plants, unicellular algae offer several experimental advantages. A relatively homogeneous population is available in unicellular algae, in contrast to land plants, in which cells differentiate into heterogeneous populations. The generation time of unicellular algae is much shorter than that required for multicellular land plants. C. reinhardtii has been the most extensively studied green alga, because it is genetically tractable. Methods for transformation have been reported, but as yet are still far from having come into practical use (Gong et al, 2011)
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