Development of a transient gene expression system in the red macroalga , Porphyra tenera

Porphyra is a commercially valuable source of food and drugs, and represents an important model organism for algal research. However, genetic research on P. tenera has been limited due to lack of a heterologous gene expression system. In the present study, we isolated a native promoter, the PtHSP70 promoter, for efficient expression of foreign genes in this organism. This promoter lies approximately 1 kb upstream of the coding sequence for Heat Shock Protein 70 (HSP70) and was isolated using adapter-mediated genomic PCR. Promoter activity was evaluated using the synthetic GUS gene (PyGUS) with optimized codons for Porphyra yezoensis. Interestingly, the PtHSP70 promoter allowed equivalent expression of PyGUS in both P. tenera and P. yezoensis, whereas the GAPDH promoter from P. yezoensis was not fully functional in P. tenera. These data suggest that the PtHSP70 promoter has a more conserved regulatory mechanism than the PyGAPDH promoter between these species. We also established an efficient transient transformation system for P. tenera by evaluating various transformation parameters such as quantity of gold particles, pressure of helium and vacuum, developmental stages of leafy gametophytes, and target distance. Under the optimal conditions of transient transformation, the frequency of GUS expression was determined by histochemical staining to be 30-50 cells per bombardment. Therefore, the new transient transformation system using the PtHSP70 promoter can be used for foreign gene expression in P. tenera, which may advance the development of P. tenera as a model organism. Introduction Porphyra, a genus of marine red macroalgae, is considered a commercially valuable source for foods, fertilizers, medicines, and chemicals (Harada et al. 1997; Oohusa 1993; Yoshizawa et al. 1995). More than 130 species of Pophyra have been reported worldwide (Zhang et al. 2005) and several species, such as P. yezoensis, P. tenera, P. seriata, and P. dentate have been cultivated in East Asia (Oohusa 1993). These species are considered model seaweeds for marine biotechnology (Fukuda et al. 2008; Oohusa 1993) because of their biological and economical importance, as well as small genome size. The haploid genomes of Porphyra are estimated to be approximately 260-500 Mbp (Kapraun et al. 1991; Le Gall et al. 1991; Matsuyama-Serisawa et al. 2007). However, heterologous gene expression to manipulate molecular pathways has been hindered by the difficulty of genetic transformation in Porphyra (Fukuda et al. 2008). Transient expression of GUS under control of the heterologous SV40 or CaMV 35S promoter has been reported in Porphyra and Gracilaria (Gan et al. 2003; Kübler et al. 1994; Kuang et al. 1998); however, the CaMV 35S promoter offered no substantial expression of GUS in P. yezoensis (Fukuda et al. 2008). Similarly, all attempts using the CaMV 35S promoter to express foreign genes in the green alga, Chlamydomonas reinhardtii, had failed (Blankenship and Kindle 1992). Possible reasons for the failure to express foreign genes using heterologous promoters in these algae may be due to the lack of the necessary regulatory elements for these promoters or poor codon-usage of the transgene. These problems were overcome by using both native promoters and codon optimization of the transgene in Chlamydomonas (Ahn et al. 2010; Franklin et al. 2002; Kozminski et al. 1993; Shao and Bock 2008). Furthermore, a recent study has reported that both codon modification and use of a native promoter enabled expression of foreign genes in P. yezoensis (Fukuda et al. 2008; Takahashi et al. 2010); synthetic GUS (PyGUS) was successfully expressed using the native promoters of both the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin genes in P. yezoensis. Because Porphyra is an important species in marine biotechnology as a model organism, it is important to develop gene manipulation systems for it. A recent study indicated success using the PyGAPDH promoter for foreign gene expression in P. yezoensis (Fukuda et al. 2008). However, it is possible that differences in regulatory elements for gene expression might exist between Porphyra species and identification of specific and universal promoters will be required for development of a foreign gene expression system in Porphyra. In the present study, we isolated the PtHSP70 promoter from P. tenera and confirmed its ability to express foreign genes in P. tenera and P. yezoensis. Our results suggest that the PtHSP70 promoter is effective for heterologous gene expression and that an optimized system for transient transformation may help promote molecular biological studies in P. tenera. Materials and Methods Culture conditions of P. tenera and P. yezoensis Leafy gametophytes of P. tenera (strain TJH-1R, Seaweed Research Center, NFRDI, Korea) grown to 2–3 mm in length were cultured further in 1 L of enriched sea life (ESL) medium at 12°C under a 10-h light/14-h dark cycle. The cultivation of leafy gametophytes of P. yezoensis strain TU-1 was also performed as described by Fukuda et al (2008). These cultures were continuously aerated with filter-sterilized air and renewed weekly. Isolation of the PtHSP70 promoter from P. tenera Genomic DNA was extracted from P. tenera using a DNeasy plant mini kit (Qiagen, USA) and polysaccharide contamination was removed using the CTAB method (Ausubel et al. 1994). Adapter ligation-mediated PCR (O'Malley et al. 2007) was adopted to amplify the promoter region of PtHSP70. Genomic DNA was digested by XhoI and then ligated with XhoI-adapter primer that had 5 ́-phosphorylation and 3 ́-amino C7. PCR was performed with adapter-ligated genomic DNA, 1.5 units of LA Taq DNA polymerase (Takara, Japan), 100 μM of each dNTP, 5 pmol of gene-specific primers (PtHSP70p R2 and R3), and adapterspecific primers (AP1 and AP2) using a T1 thermal cycler (Biometra, Germany). The genespecific primers were designed using a consensus sequence from P. seriata and P. yezoensis ESTs. Amplification was performed with 35 cycles at 94°C for 30 s, 62°C for 1 min, and 72°C for 4 min. Primers used for isolation of the PtHSP70 promoter are listed in Table 1. For isolation of the 5 ́-promoter region of PtHSP70, genomic DNA was digested using BamHI and adapter ligation, and a second PCR amplification was performed using promoter-specific primers (PtHSP70p R4 and R5) and adapter-specific primers (Table 1). Finally, the PtHSP70 promoter (1030-bp) was amplified by PCR with the Pt-HSP70-P-1kF and PtHSP70p R3 primers designed from the combined sequences of two clones obtained from the first and second PCRs. The nucleotide sequence of PtHSP70 promoter was shown in Fig. 1. Plasmid construction for transient PyGUS expression The vector backbone used in the present study was obtained from the GAPDH-PyGUS construct (Fukuda et al. 2008). The PyGAPDH promoter was replaced with the PtHSP70 promoter and three constructs were generated to evaluate expression levels of PyGUS. Plasmid PtHSP70-PyGUS1 (Fig. 2) contained the PtHSP70 promoter upstream of PyGUS. For PtHSP70-PyGUS2, a 3 ́-modification of the PtHSP70 promoter was performed by inserting a 5 ́-coding sequence (18-bp) of PtHSP70. The 5 ́-coding sequence (21-bp) of PyGAPDH was inserted 3’ of the PtHSP70 promoter to construct PtHSP70-PyGUS3. Restriction enzyme sites used for the three constructs are displayed in Fig. 2. The following primers were used for generating these constructs: PtHSP70-GUS1, 5 ́-AAG GAT CCC ATC GTC GGG TGC ACA-3 ́ and 5 ́-AAG GAT CCC ATC GTC GGG TGC ACA-3 ́; PtHSP70GUS2, 5 ́-TTG GAT CCG CTC ACT GCA GAC GCC AT-3 ́ and 5 ́-TTG GAT CCG CTC ACT GCA GAC GCC AT-3 ́; PtHSP70GUS3, 5 ́-AGA CGC CAT GGT CGG GTG CAC A-3 ́ and 5 ́-AGA CGC CAT GGT CGG GTG CAC A-3 ́. Transient gene expression using particle bombardment The expression constructs recovered from E. coli cells using a Plasmid® midi kit (Qiagen, USA). Transient transformationwas performed by particle bombardment using 60 mg gold particles (0.6 μm diameter). Particles were washed with 1 ml of 70% ethanol by vortexing, rinsed three times with sterile water, and then resuspended in a 50% glycerol solution. Different amounts of gold particles (25, 50, 75, 100, 250, and 500 μg) coated with 20 μg plasmid were used to evaluate efficiency of transient transformation, according to the method of Fukuda et al (2008). Leafy gametophyte samples on microfiber filter (25-mm diameter, GF/B; Whatman, Germany) were placed in 10 cm Petri dishes filled with ESL 0.6% agar medium. Particle bombardment was performed using a particle delivery system (PDS, 1000/He; BioRad, USA) under various pressure conditions (900, 1000, and 1300 psi of helium, 28 in Hg of vacuum). Different target distances (3, 6, and 9 cm) were also tested. After bombardment, samples were incubated in liquid ELS media at 12°C for 2 days in the dark. Transient transformation of P. yezoensis was performed as previously described (Fukuda et al. 2008), other than the particle bombardment. DNA transfer was carried out using a PDS 1000/He under these conditions: 900 psi of helium, 28 in Hg of vacuum, 3 cm of target distance, and 150 g gold particles/shot. Histochemical and fluorometric GUS assays GUS histochemical staining using 5-bromo-4-chloro-3-indolylglucuronide (X-gluc; Sigma, USA) was carried out according to Fukuda et al (2008). Quantitative fluorometric assays for GUS activity were performed on gametophytes 48h after bombardment according to Jefferson, et al. (1987), using p-nitrophenyl glucuronide (PNPG; Sigma, USA) for P. tenera and 4-methylumbelliferyl-D-glucuronide (4-MU; Calbiochem, Germany) for P. yezoensis. For P. yezoensis, GUS values were expressed as pmoles of 4-MU per minute per milligram protein. The fluorescence was measured with a spectrofluorometer (Picofluor; Turner Designs, USA). Protein concentrations were determined by the method of Bradford (1976). Because GUS activity in transiently transformed P. tenera was too low to measure fluoromet