A codon-optimized bacterial antibiotic gene used as selection marker for stable nuclear transformation in the marine red alga Pyropia yezoensis.

Marine macroalgae play an important role in marine coastal ecosystems and are widely used as sea vegetation foodstuffs and for industrial purposes. Therefore, there have been increased demands for useful species and varieties of these macroalgae. However, genetic transformation in macroalgae has not yet been established. We have developed a dominant selection marker for stable nuclear transformation in the red macroalga Pyropia yezoensis. We engineered the coding region of the aminoglycoside phosphotransferase gene aph7″ from Streptomyces hygroscopicus to adapt codon usage of the nuclear genes of P. yezoensis. We designated this codon-optimized aph7″ gene as PyAph7. After bombarding P. yezoensis cells with plasmids containing PyAph7 under the control of their endogenous promoter, 1.9 thalli (or individuals) of hygromycin-resistant strains were isolated from a 10-mm square piece of the bombarded thallus. These transformants were stably maintained throughout the asexual life cycle. Stable expression of PyAph7was verified using Southern blot analysis and genomic PCR and RT-PCR analyses. PyAph7 proved to be a new versatile tool for stable nuclear transformation in P. yezoensis.

Molecular characterization and expression analysis of sodium pump genes in the marine red alga Porphyra yezoensis.

Sodium pumps (EC 3.6.3.9, Na(+)-ATPase), which mediate excretion of Na(+) from the cell, play a crucial role in Na(+) homeostasis in eukaryotic cells. The objective of this study is to understand the Na(+) efflux system in a marine red alga. We identified a novel sodium pump gene, PyKPA2, from the marine red alga Porphyra yezoensis. The amino acid sequence of PyKPA2 shares 65 % identity with PyKPA1, a previously identified P. yezoensis sodium pump. Similar to PyKPA1, PyKPA2 contains conserved sequences for functions such as phosphorylation, ATP binding, and cation binding. Phylogenetic analysis revealed that the two genes cluster with sodium pumps from algae. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that PyKPA1 is expressed preferentially in sporophytes, whereas PyKPA2 is expressed specifically in gametophytes. RT-PCR and quantitative real-time PCR analysis revealed that PyKPA1 and PyKPA2 transcripts were upregulated and downregulated, respectively, in gametophytes during exposure to alkali stress. In addition, transcription of both genes in gametophytes was also induced by cold stress. These results suggest that PyKPA1 and PyKPA2 play an important role in alkali and cold stress tolerance.

Heterologous activation of the Porphyra tenera HSP70 promoter in Bangiophycean algal cells.

Porphyra has attracted great attention for its biological and industrial importance. However, establishment of a stable nuclear transformation has not yet been achieved in these organisms, which impedes the molecular biological study and the development of a molecular breeding method for them. Toward establishing the stable transformation, we have recently developed an efficient transient gene expression system in Bangiophycean algae, in which the HSP70 promoter from P. tenera (PtHSP70 promoter) was activated heterologously in P. yezoensis cells. Since heterologous promoters are required for homologous recombination-based stable transformation, the identification of heterologously activated promoters is important in establishing a stable transformation system in individual Bangiophycean alga. We here examined the activation of the PtHSP70 promoter using the GC-rich PyGUS reporter system in additional Porphyra and Bangia species. The results indicated that this promoter drove expression of the PyGUS gene efficiently in all examined algae, whereas there was quite low expression of PyGUS by the cauliflower mosaic virus 35S promoter that is widely used as a heterologous promoter in the transformation of green land plants. Therefore, heterologous activation of the PtHSP70 promoter could promote the establishment of the stable transformation system in various kinds of Bangiophycean algae.

Transient gene expression system established in Porphyra yezoensis is widely applicable in Bangiophycean algae.

The establishment of transient gene expression systems in the marine red macroalga Porphyra yezoensis has been useful for the molecular analysis of cellular processes in this species. However, there has been no successful report about the expression of foreign genes in other red macroalgae, which has impeded the broader understanding of the molecular biology of these species. We therefore examined whether the P. yezoensis transient gene expression system was applicable to other red macroalgae. The results indicated that a codon-optimized GUS, designated PyGUS, and plant-adapted sGFP(S65T) were successfully expressed under the control of the P. yezoensis PyAct1 promoter in gametophytic cells of six Porphyra species and also in Bangia fuscopurpurea, all of which are classified as Bangiophyceae. In contrast, there were no reporter-expressing cells in the Florideophycean algae examined. These results indicate the availability of PyGUS and sGFP as reporters and the 5' upstream region of the PyAct1 gene as a heterologous promoter for transient gene expression in Bangiophycean algae, which could provide a clue to the efficient expression of foreign genes and transformation in marine red macroalgae.