Identification and Efficient Utilization of Antibiotics for the Development of a Stable Transformation System in Porphyra yezoensis (Bangiales, Rhodophyta)

Abstract

Despite the development of transient gene expression in red algae Porphyra species, a stable transformation system has not been established in these algae yet. One reason for the difficulty in the transformation is the lack of a way to select transformed cells from the algae. Thus, to identify antibiotics suitable for the selection of stably transformed cells, we tested the gametophyte for sensitivity to 6 antibiotics, ampicillin (Am), kanamycin (Km), hygromycin (Hm), geneticin (G418), chloramphenicol (Cm) and paromomycin (Pm), which are frequently employed as selection agents in the transformation of land plants and microalgae. Sensitivity tests, which were carried out with different concentrations (0-10 mg mL-1) of antibiotics, showed that Hm, G418, Cm and Pm exhibited a lethal effect at more than 1.0 mg mL-1, suggesting the suitability of these four antibiotics for the selection of genetically transformed cells from P. yezoensis gametophytes, while P. yezoensis gametophytes were highly resistant to Am and Km. We also succeeded in reducing the antibiotic concentrations by lowering the pH value and salt concentration using an artificial synthetic medium. Increasing the sensitivity of gametophytic cells to Hm, G418, Cm and Pm in the modified synthetic medium will contribute to establishing a genetic transformation system in P. yezoensis

Current Advances in Seaweed Transformation

“…Therefore, the next step is to develop the gene targeting system via integration of a foreign gene into the genome and the system for selection of transformed cells. Since candidates of antibiotic agents for selection of transformed algal cells were mentioned recently [101][102][103]106], it is necessary to confirm the possibility of stable integration of a plasmid or a DNA fragment containing the selection maker gene into the seaweed genome. Once a positive result is obtained, it could lead us to establish the gene targeting method via the homologous recombination using an appropriate antibiotics resistance gene, if possible, with the heterologous promoter.…”

“…Recently, the sensitivity of P. yezoensis gametophytes to ampicillin, kanamycin, hygromycin, geneticin (G418), chloramphenicol and paromomycin was investigated, and lethal effects of these antibiotics on gametophytes were observed at more than 2.0 mg mL -1 of hygromycin, chloramphenicol and paromomycin and 1.0 mg mL -1 of G418, whereas P. yezoensis gametophytes were highly resistant to ampicillin and kanamycin [101]. Although these concentrations are in fact very high in comparison with the cases for the red alga Griffithsia japonica and the green alga C. reinhardtii that were highly sensitive to 50 μg mL -1 and 1.0 μg mL -1 of hygromycin [96,102], these four antibiotics and corresponding resistance genes are suitable for the selection of genetically transformed cells from P. yezoensis gametophytes.…”

“…Insensitivity to hygromycin is inconsistent with the case for red and brown seaweeds [101][102][103]106]. It is therefore necessary to check the sensitivity of green seaweed cells to other antibiotics to identify the genes employable for selection of transformed cells, which could stimulate the development of the genetic transformation system in green seaweeds.…”

A technical breakthrough close at hand: feasible approaches toward establishing a gene-targeting genetic transformation system in seaweeds

“…The findings above indicate that using antibiotics will be pragmatic. Doses of 2.0 mg/ml hygromycin, chloramphenicol, or paromomycin, or of 1.0 mg/ml G418, were lethal to P. yezoensis gametophytes, although these gametophytes were also highly resistant to ampicillin and kanamycin (Takahashi et al, 2011 ). Thus, as mentioned in Mikami ( 2013 ), these four antibiotics and their corresponding resistance genes are thought to be suitable for the selection of genetically transformed cells from P. yezoensis gametophytes.…”

A Codon-Optimized Bacterial Antibiotic Gene Used as Selection Marker for Stable Nuclear Transformation in the Marine Red Alga Pyropia yezoensis

“…Recently, we have revealed that P. yezoensis cells are sensitive to several aminoglycoside antibiotics, including hygromycin B, paromomycin, and geneticin (Takahashi et al 2011). Thus, these antibiotics are possible candidate selection agents for stable P. yezoensis transformation.…”