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.


Introduction
The marine red macroalga Pyropia yezoensis (nori in Japanese) is one of the most important marine crop. It is widely cultivated in eastern Asian countries, including Japan, Korea, and China, and generates US$1.3 billion per year (Blouin et al. 2011). In addition, P. yezoensis has attracted considerable interest as a model for physiological and genetic studies of marine red algae (Saga and Kitade 2002;Waaland et al. 2004). To date, several studies have been performed to make this alga a sophisticated model organism. For example, a laboratory culturing system in which the life cycle of P. yezoensis could be completed within a few months was established (Kuwano et al. 1996). In addition, a database for expressed sequenced tags (EST) analysis is now available (Nikaido et al. 2000;Asamizu et al. 2003) and recently, the draft data of whole genome sequence has been analyzed by next generation sequencing (Nakamura et al. 2013). However, a stable transformation system, a powerful tool both for elucidating gene functions and conferring valuable characteristics to an organism, has not yet been established for P. yezoensis or other marine macroalgae.
As an initial step in establishing stable transformation, we previously developed a transient gene expression system to monitor gene expression in P. yezoensis cells using particle bombardment. Because P. yezoensis genes have a strong GC bias in the third nucleotide of their codons, it is important to adapt codon usage of foreign genes to the nuclear genes of P. yezoensis for their efficient expression. In fact, codonoptimized β-glucuronidase (PyGUS) and GC-rich fluorescent proteins, such as AmCFP and sGFP(S65T), have been expressed in P. yezoensis cells under the control of an endogenous promoter (Fukuda et al. 2008;Mikami et al. 2009;Uji et al. 2010 In addition to developing an efficient expression system, a reliable method to select and isolate transformed cells is required to establish stable transformation in macroalgae. Recently, we have revealed that P. yezoensis cells are sensitive to several aminoglycoside antibiotics, including hygromycin B, paromomycin, and geneticin . Thus, these antibiotics are possible candidate selection agents for stable P. yezoensis transformation. Regarding a selection marker, the aminoglycoside phosphotransferase gene aph7″ from Streptomyces hygroscopicus , which confers resistance against hygromycin B, should be available for hygromycin-based stable transformant selection because the GC content in its coding region is as high as 70.94 % (Zalacain et al. 1986). The aph7″ gene has been successfully used for the transformation of green microalgae, such as Chlamydomonas reinhardtii (Berthold et al. 2002), whose codons are also rich in GC residues. However, several codons that are rarely used in P. yezoensis nuclear genes are found in the aph7″ gene, especially in its N-terminal region. These mismatches in codon usage would be predicted to inhibit efficient translation in P. yezoensis cells.
Thus, in the present study, we synthesized a codonoptimized aph7″ gene and examined its utility as a selection marker for stable nuclear transformation in P. yezoensis.

Culturing of P. yezoensis
Gametophytes of P. yezoensis strain TU-1 and transformants were cultured in enriched sea life (ESL) medium under conditions described by Fukuda et al. (2008).

Plasmid Construction
To construct a pEA7 plasmid, a fragment containing the ORF of PyAph7 and 3′ UTR of CrRbcS2 was amplified using pHyg4 as a template and a pair of primers, XbaI-PyAph7-F (5′-GCTCTAGAATGACGCAGGAGTCCCTGCTGCT GCTC-3′) and Eco RI-CrRbcS2-R (5′-GGAATTCTTCCA TGGGATGACGGGCCCGG-3′). The amplified PCR product was digested with XbaI and EcoRI and subsequently inserted into Xba I-Eco RI-digested p35S-PyGUS (Fukuda et al. 2008), which was designated p35S-PyAph7. To replace the CaMV 35S promoter with an endogenous promoter, the 5′ upstream region of PyElf1 was amplified using pPyElf1-PyGUS ) as a template and the following primers: Hin dIII-PyElf1-F/XbaI-PyElf1-R (5′-CCCAA G C T T C C A G A C C C G T G G A A A G TA C C AT C -3 ′ / 5 ′ GCTCTAGACTTGCCCATGGTGGGGGGG-3′). The PCR product was digested with HindIII and XbaI and subsequently inserted into the Hin dIII-Xba I site of p35S-PyAph7. This resulted in pEA7 construction.

Particle Bombardment
Expression plasmids were purified from 100 mL of Escherichia coli culture using a NucleoBond Xtra Midi (MACHEREY-NAGEL, Germany). For particle bombardment, a gametophytic thallus with monosporangia covering a wide range of the thallus (>10 mm in width) was cut into 10-mm square pieces, which were subsequently placed on a filter paper. After removing excess fluid, the expression plasmids were introduced into the gametophytic cells using PDS-1000/He particle bombardment under the conditions described previously ).

Isolation of Hygromycin-Resistant Transformants
The bombarded algal pieces were cultured in a 100-mL glass flask (Iwaki Sci Tech Div., Asahi Techno Glass, Japan) in 50 mL of ESL medium under non-selective conditions for 1 week. Subsequently, the medium was replaced with the ESL medium containing hygromycin B (final concentration of 1 mg mL −1 ), and the medium was renewed weekly. After incubation for 6-8 weeks in the antibiotic-containing medium, visible hygromycin-resistant transformants regenerated from the pieces of bombarded thalli were individually isolated in another culture flask and continuously cultured in ESL medium with or without 1 mg mL −1 hygromycin B.

Assay for Hygromycin Resistance
To prepare individuals for hygromycin resistance assay, gametophytes isolated as hygromycin-resistant transformants were clonally propagated for 3 weeks in different culture flasks containing ESL medium via monospores. Gametophytes of transformants or wild-type strains (ca. 20 mm in length) cultured were respectively transferred into a 6-well plate (3 individuals/well) (Iwaki Sci Tech Div., Asahi Techno Glass, Japan) containing 5 mL of ESL medium with 0, 1.0, 2.5, 5.0, 7.5, or 10.0 mg mL −1 hygromycin B and incubated under shaking culture for 2 weeks at 15°C. The medium was renewed weekly. After culture, gametophyte viability was estimated by staining using 0.01 % erythrosine (Wako Pure Chemical Industries, Japan) in ESL medium according to a previous report .

Genomic PCR and RT-PCR
Genomic DNA was extracted from gametophytes of transformants cultured for 4 weeks in ESL medium without hygromycin B or wild-type strains for genomic PCR as described by Hwang et al. (2010) and purified using a phenol-chloroform extraction and ethanol precipitation. The precipitate was resuspended in 50 μL of TE buffer, and 2 μL of this suspension was used as a template for genomic PCR. RNA extraction and cDNA synthesis for RT-PCR were performed as described by Uji et al. (2012). Genomic PCR and RT-PCR analyses were conducted using TaKaRa LA Taq with GC buffer (TaKaRa-Bio). The primer pairs PyAph7-RT-F/R (5′-CATTGACTCGGACGACTCCTACGCGAG-3′ /5′ -AAGTCGTGCAGGAAGGTGAAG-3′) and PyElf1-RT-F/R (5′-AAGGCCAAGGCACCCAAGCTG-3′/5′-ACCA CACCAAGAGCGTCCAATC-3′) were used to amplify the fragments of PyAph7 (864 bp) and PyElf1 (734 bp), respectively. The amplified PCR products were examined on a 1.3 % agarose gel.

Southern Blotting
Genomic DNA was extracted using the cetyl trimethyl ammonium bromide (CTAB) method from 1.0 g (FW) of wild type and transformants that had been cultured for more than 3 months after isolation. Extracted DNA was further purified by ultracentrifugation as follows: 3.1 g of cesium chloride (CsCl) and 15 μL of ethidium bromide (EtBr) were added to 3.0 mL of the DNA solution and centrifuged at 400,000×g for 24 h at 20°C. The DNA band was visualized under UV light and collected. EtBr was removed by three extractions with an equal volume of 1-butanol. CsCl was removed by ethanol precipitation three times. Purified DNA (2.0 μg) was digested with Pst I, run on agarose gel, and transferred to a nylon membrane. The pEA7 plasmid was digested with XbaI and SalI and a 730-bp fragment of PyAph7 was collected. This fragment was labelled by random 32P priming and used as a probe (see Fig. 1a).

Results and Discussion
To optimize the codon usage of the aph7″ coding region to that of P. yezoensis, we employed site-directed mutagenesis using a pHyg4 plasmid that contained the aph7″ gene (Berthold et al. 2002) and a KOD-Plus-Mutagenesis Kit (Toyobo, Japan) with the oligonucleotides shown in Table S1. The synthetic aph7″ gene was designated PyAph7 (Fig. S1). Subsequently, the protein coding region of PyAph7 was fused with the endogenous PyElf1 promoter ). The resulting plasmid was designed pEA7 (Fig. 1a).
The pEA7 expression plasmids containing PyAph7 were introduced into P. yezoensis gametophytes by particle bombardment. When gametophytic thalli with the introduced pEA7 were cultured in a non-selective ESL medium for 1 week (Fig. 1b), some monospores (asexual spores) were released from the bombarded thalli and adhered to the bottom of the culture flask (Fig. 1c). The medium was subsequently replaced with selective ESL medium containing 1.0 mg mL −1 hygromycin B, which effectively kills wild-type cells or, at least, completely inhibits their growth . After 6-8 weeks of culture in the selective medium, hygromycin-resistant thalli from them released monospores or from vegetative cells of bombarded blades were approximately 5-10 mm long (Fig. 1d, e). These transformants were transferred into separate culture vessels to identify the homogeneous lines and cultured further in a non-selective ESL medium (Fig 1b). Consequently, an average of 1.9 thalli (or individuals) of the hygromycin-resistant strains were obtained from a piece of the bombarded thallus (29 transformants per 15 bombardments). These transformants were successfully Finally, to verify whether the exogenous PyAph7 gene had been successfully introduced and expressed in these isolated transformants, genomic PCR and RT-PCR analyses were performed on four transformant strains (EA1-EA4). Using primers specific for the PyAph7 gene sequence, a DNA fragment of the expected size was amplified for all of the examined strains, whereas this fragment was not observed in a wildtype strain (Fig. 3a). This indicated that PyAph7 had been successfully introduced and expressed in these transformants. We further analyzed the stable integration of PyAph7 into the genome by Southern blot analysis. The Southern blot analysis revealed that multiple DNA fragments could be detected in all of the examined transformants, which indicated that PyAph7 had multiplied and randomly integrated into the genome (Fig. 3b)  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 fragment that corresponded to the full length of the pEA7 vector were commonly detected in all transformed strains. The results intimate the possibility that a part of introduced pEA7 vector might be stably maintained as entire circular plasmids in the transformed cells through cell divisions and propagation. Interestingly, there have been several reports on plasmid DNA isolation from some red macroalgae, including Pyropia tenera, a closely related species of P. yezoensis (Goff and Coleman 1990;Choi et al. 2000Choi et al. , 2001. Thus, we need to investigate on mechanisms for maintaining plasmid DNA in P. yezoensis cells. Taken together, the codon-optimized PyAph7 introduced by particle bombardment was stably maintained and expressed in P. yezoensis and conferred resistance to hygromycin B. Our results demonstrate that PyAph7 is useful as an efficient selection marker for stable nuclear transformation of P. yezoensis . This is the first report of a bacterial antibiotic gene used as a selection marker for stable transformation in marine macroalgae. Further development of this stable transformation system in P. yezoensis will overcome some of the barriers in molecular biological studies of marine red algae.