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

Abstract

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.

Fig. 1

Isolation of hygromicin-resistant transformants in P. yezoensis. a Schematic diagram of the hygromycin selective vector pEA7. The coding region of PyAph7 is fused in-frame to 5′ PyElf1 (promoter, 5′-untranslated region of PyElf1 and the initiation codon). 3′ CrRbcS2 indicates the 3′ untranslated region of the RbcS2 gene from Chlamydomonas reinhardtii. The position and length of the DNA fragment amplified by genomic PCR or RT-PCR are indicated. The position of the probe used in Southern blotting is indicated (probe). b Timeline for isolating hygromicin-resistant transformants (hyg B, hygromycin B). c Macroscopic view of the bottom of the culture flask on which monospores released from bombarded thalli were attached (arrowheads). Scale bar = 10 mm. d Hygromycin-resistant thalli regenerated from monospores attached to the bottom of a culture flask (arrowheads). Scale bar = 10 mm. e Hygromycin-resistant thalli regenerated from the vegetative cells of a bombarded thallus (arrowheads). Scale bar = 5 mm

Fig. 2

Analysis of hygromycin B resistance for wild-type and transgenic P. yezoensis strains. Survival rates of wild type and six lines of the hygromycin-resistant strains (EA1–EA6) when cultured with varying concentrations of hygromycin B (1.0–10.0 mg mL⁻¹). The survival rate was calculated by counting viable and dead gametophytes during 2 weeks culture in ESL medium containing hygromycin B. Values are means ± SDs (n = 30)

Fig. 3

PCR and Southern blot analyses of hygromicin-resistant transformants. a Expression of the exogenous PyAph7 gene in hygromycin-resistant transformants was detected by genomic PCR and RT-PCR. PCR was performed using primers specific for the PyAph7 gene sequence (Fig. 1) and genomic DNA or total RNA from a wild-type strain and the transformants EA1–EA4. PyElf1 was used as the internal control gene in P. yezoensis. Only transformants were expected to yield an 864 bp fragment of PyAph7. b Southern blot analysis of hygromycin-resistant transformants. Genomic DNA from a wild-type strain and the transformants EA1–EA4 were digested with PstI, separated on agarose gel, transferred to nylon membrane, and hybridized with a labelled probe corresponding to the PyAph7 fragment (Fig. 1). Lane M, molecular weight marker