Chloramphenicol acetyltransferase—a new selectable marker in stable nuclear transformation of the red alga Cyanidioschyzon merolae

Abstract

In this study, we have shown the applicability of chloramphenicol acetyltransferase as a new and convenient selectable marker for stable nuclear transformation as well as potential chloroplast transformation of Cyanidioschyzon merolae-a new model organism, which offers unique opportunities for studding the mitochondrial and plastid physiology as well as various evolutionary, structural, and functional features of the photosynthetic apparatus.

Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments

“…In case of other antibiotics, commonly used as chloroplast transformation markers, the rate of spontaneous resistance can be very high and even similar to the rate of chloroplast transformation (Day and Goldschmidt-Clermont 2011). As argued before (Zienkiewicz et al 2015), the cat gene requires extensive nucleotide optimization. The codon usage analysis showed significant differences between nuclear and plastid codon frequencies, prompting a specific sequence optimization, to the most frequently occurring codons in the C. merolae plastid.…”

“…The codon-optimization of the cat gene sequence was performed in accordance with the method described earlier (Zienkiewicz et al 2015). The chloroplast-optimized cat gene sequence (GenBank KX056487), annotated cat CH, revealed 80% identity with the native cat gene (Tn9 from pACYC184 vector).…”

“…by application of an herbicide resistance gene (Lapidot et al 2002). Recently, we have shown that a codon-optimized sequence of the cat gene, coding for chloramphenicol acetyltransferase can be used as a selectable marker for stable nuclear but also, potentially chloroplast transformation of C. merolae (Zienkiewicz et al 2015). Here, we set out to establish the first protocol for stable chloroplast transformation of C. merolae , allowing us to perform a stable, site-directed mutagenesis of the plastid genome in the future.…”

“…In the following studies we have utilized the efficient expression of chloroplast encoded proteins to achieve high enough level of chloramphenicol acetyltransferase protein production, capable of providing sufficient protection of chloroplast ribosome machinery from chloramphenicol (Zienkiewicz et al 2015), even in the apparently unfavorable environment of chloroplasts. The problem of the exogenous proteins instability, expressed in chloroplast has been encountered before (Birch-Machin et al 2004; Sakamoto 2006), however little explanation has been given.…”

“…Additionally, C. merolae doesn’t possess the Dicer enzyme (Casas-Mollano et al 2008), therefore the RNAi phenomenon doesn’t occur (Day and Goldschmidt-Clermont 2011). It has been presented in our previous work, that a stable nuclear transformant line with the cat gene fused with a signal peptide, directing the chloramphenicol acetyltransferase to the chloroplast, had allowed this organism’s cells to grow under high selective pressure of the antibiotic (Zienkiewicz et al 2015). Therefore, we decided that the marker gene of our choice will be chloramphenicol acetyltransferase (CAT).…”