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Current Advances in Seaweed Transformation

Written By

Koji Mikami

Submitted: March 1st, 2012 Published: July 1st, 2013

DOI: 10.5772/52978

An Integrated View of the Molecular Recognition and Toxinology IntechOpen
An Integrated View of the Molecular Recognition and Toxinology From Analytical Procedures to Biomedical Applications Edited by Gandhi Radis-Baptista

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An Integrated View of the Molecular Recognition and Toxinology

Edited by Gandhi Rádis Baptista

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1. Introduction

Frederick Griffith reported the discovery of transformation in 1928 [1]. Since a harmless strain of Streptococcus pneumoniae was altered to a virulent one by exposure to heat-killed virulent strains in mice, Griffice hypothesized that there was a transforming principle in the heat-killed strain. It took sixteen years to indentify the nature of the transforming principle as a DNA fragment released from virulent strains and integrated into the genome of a harmless strain [2]. Such an uptake and incorporation of DNA by bacteria was named transformation. Remarkably, an epoch-making technology in the form of artificial transformation protocol for the model bacterium Escherichia coli was established by Mandel and Higa in 1970 [3], which stimulated the development of artificial genetic transformation systems in yeasts, animals and plants. In plants, genetic transformation is a powerful tool for elucidating the functions and regulatory mechanisms of genes involved in various physiological events, and special attention has been paid to plant improvements affecting food security, human health, the environment and conservation of biodiversity. For instance, researchers have focused on the creation of organisms that efficiently produce biofuels and medically functional materials or carry stress tolerance in the face of uncertain environmental conditions [4-6].

Although the first success in the creation of transgenic mouse was carried out by injecting the rat growth hormone gene into a mouse embryo in 1982 [7], the protocol for artificial genetic transformation in plants was established earlier than that in animals. Following the discovery of the soil plant pathogen Agrobacterium tumefaciens, which is responsible for producing plant tumors, in 1907 [8], it was found that the tumor-inducing agent is the Ti plasmid containing T-DNA, a particular DNA segment containing tumor-producing genes that are transferred into the nuclear genome of infected cells [9]. By replacing tumor-producing genes by a gene of interest within the T-DNA region, infection of A. tumefaciens carrying a modified Ti plasmid results in insertion of a DNA fragment containing the desired genes into the genomes of plants by genetic recombination. Since the report of this protocol in the early 1980s [10,11], transformation mediated by A. tumefaciens has become the most commonly used method to transmit DNA fragment into higher plants [12].

Since not all plant cells are susceptible to infection by A. tumefaciens, other methods were developed and are available in plants. Particle bombardment [13], which is also referred to as microprojectile bombardment, particle gun or biolistics, makes use of DNA-coated gold particles, which enables the transient and stable transformation of almost any type of cell, regardless of rigidity of the cell wall, and is thus extensively used for land plants. For protoplasts, electroporation is well employed, for which a high-voltage electrical pulse temporarily disturbs the phospholipid bilayer of the plasma membrane, allowing cells to take up plasmid DNAs [14,15]. In addition, the polyethylene glycol (PEG)-mediated transformation system is also thought to affect the plasma membrane and induce the uptake of DNAs into cells [15,16] and is almost exclusively applied with the moss Physcomitrella patens and liverwort Marchantia polymorpha [17,18]. Therefore, several kinds of genetic transformation methods are now available in land green plants.

Seaweeds are photosynthetic macroalgae, the majority of which live in the sea, and are usually divided into green, red and brown algae. Traditionally, all classes of seaweeds are known as human foods especially in Asian countries; for instance, red algae are known as Nori and brown algae are called Konbu and Wakame in Japan. In addition, red and brown algae are utilized as the sources of industrially and medically valuable compounds such as phycoerythrin, n-3 polyunsaturated fatty acids, porphyran, ager and carrageenan from red algae, and fucoxantine, fucoidan and alginate from brown algae [19-22]. Thus, to make new strains carrying advantageous characteristics benefiting industry and medicine, researchers have worked hard since the early 1990s to establish methods of genetic transformation in seaweeds [20,23,24]. However, the process is very difficult, and most of the early studies were reported in conference abstracts without the accompanying manuscript publication [25-28]. This situation has hampered us from gaining an understanding of gene functions in various physiological regulations and also a utilization of seaweeds in biotechnological applications.

Transformation can be divided into genetic (stable) and transient transformations under the control of the genes introduced into cells. In genetic transformation, genes introduced by genetic recombination are maintained in the genome through generations of cells, whereas in transient transformation, rapid loss of introduced foreign genes is usually observed. Establishing the genetic transformation system requires four basal techniques: an efficient gene transfer system, an efficient expression system for foreign genes, an integration and targeting system to deliver the foreign gene into the genome, and a selection system for transformed cells. It is notable that the transient transformation system is completed by the first two of the four required systems. In this respect, the development of an efficient and reproducible transient transformation system is the most critical step to establishing a genetic transformation system in seaweeds.

The current progress in establishing of both transient and genetic transformation systems in macroalgae is reviewed here. Although high-quality review articles for algal transformation have been published previously [20,23,24], I believe addressing the recent activity in seaweed transformation provides valuable information for seaweed molecular biologists and breeding scientists. Since considerable technical improvement was recently made in red seaweeds [29,30], I focus here on the current progress in red algal transient transformation with summarizing pioneer and recent studies related to seaweed genetic transformation.

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2. Transformation in red seaweeds

2.1. Pioneer studies for transient transformation

As far as I know, Donald P. Cheney is the pioneer in researching red algal transformation. He and his colleague performed transient transformation of the red alga Kappaphycus alvarezii using particle bombardment [25], which was the first report about the transient transformation of seaweeds (Table 1). In this case, the Escherichia coli uidA gene encoding β-glucuronidase (GUS) was expressed as a reporter under direction of the cauliflower mosaic virus (CaMV) 35S promoter (CaMV 35S-GUS gene). Since the GUS expression can be visualized as a blue color following treatment with X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) and also be quantified by fluorometric analysis [31,32], this reporter gene is widely used in land green plants having no background of the GUS activity [33,34]. In addition, the CaMV 35S promoter is heterologously used in land green plants as a strong constitutive and non-tissue-specific transcriptional regulator [35,36]. Therefore, it is a natural choice for the selection of the CaMV35S-GUS gene by pioneers for initial trials of seaweed transformation.

To date, studies have been mainly focused on Porphyra species because of their economical values. As shown in Table 1, expression of the CaMV 35S-GUS gene was previously observed in P. miniata, P. tenera and P. yezoensis [37-42], all of which were performed by electoroporation using protoplasts. Kuang et al. [38] also tested the particle bombardment of the CaMV 35S-GUS gene in P. yezoensis and got positive results. Moreover, the availability of mammalian-type simian virus 40 (SV40) promoter was reported to express the E. coli lacZ reporter gene, encoding β-galactosidase cleaving colorless substrate X-gal (5-bromo-4-chloro-3-indolyl-β-galactopyranoside) to produce a blue insoluble product [43], in P. haitanensis, Gracilaria chagii and K. alvarezii by electroporation or particle bombardment [44,45].

2.2. Recent improvement of the transient transformation system in Porphyra

As noted above, pioneer experiments of red algal transient transformation were performed using plant viral CaMV 35S RNA and animal viral SV40 promoters in combination with GUS and lacZ reporter genes (Table 1). The CaMV 35S and SV40 promoters are typical eukaryotic class II promoters with a TATA box and thus are generally employed to drive transgenes in dicot plant and animal cells, respectively [46,47]. However, we have found that the TATA box is not usually found in the core promoters of P. yezoensis genes (unpublished observation), and we thus proposed that there were differences in the promoter structure and transcriptional regulation of protein-coding genes between red algae and dicot plants. Indeed, we recently observed quite low activity of the CaMV 35S promoter and the GUS reporter gene in P. yezoensis gametophytec cells [29,30,48]. These observations are completely opposite from the results in previous reports using the CaMV 35S promoter [25,37-41]. As a result, the transient transformation system in red seaweeds has recently been improved by resolving this problem.

Species Status of expression Gene transfer method Promoter Marker or Reporter Ref.
Kappaphycus alvarezii transient particle bombardment CaMV 35S GUS [25]
Porphyra miniata transient electroporation CaMV 35S GUS [37]
Porphyra yezoensis transient Electroporation
particle bombardment		 CaMV 35S GUS [38]
Porphyra tenera transient electroporation CaMV 35S GUS [39]
Porphyra yezoensis transient electroporation rbcS GUS [40]
Porphyra yezoensis transient electroporation CaMV 35S GUS [41]
Porphyra yezoensis transient electroporation CaMV 35S
β-tubulin		 GUS [42]
Gracilaria changii transient particle bombardment SV40 lacZ [44]
Porphyra haitanensis transient SV40 CAT [128]
Porphyra yezoensis transient electriporation SV40 CAT, GUS [129]
Porphyra yezoensis transient electroporation Rubisco GUS, sGFP(S65T) [130]
Porphyra yezoensis transient particle bombardment CaMV 35S
PyGAPDH		 PyGUS [48]
Porphyra yezoensis transient particle bombardment PyAct1 PyGUS [66]
Porphyra yezoensis transient particle bombardment PyAct1 AmCFP [70]
Porphyra yezoensis transient particle bombardment PyAct1 AmCFP, ZsGFP, ZsYFP, sGFP(S65T) [71]
Porphyra tenera
Porphyra yezoensis		 transient particle bombardment PtHSP70
PyGAPDH		 PyGUS [85]
Porphyra species*
Bangia fuscopurpurea		 transient particle bombardment PyAct1 PyGUS
sGFP(S65T)		 [86]
Porphyra species*
Bangia fuscopurpurea		 transient particle bombardment PtHSP70 PyGUS [87]
Porphyra yezoensis stable Agrobacterium-mediated
gene transfer		 CaMV 35S GUS [26]
Porphyra leucostica stable ekectroporation CaMV 35S lacZ [27]
Porphyra yezoensis stable Agrobacterium-mediated
gene transfer		 (unknown) (unknown) [28]
Kappaphycus alvarezii stable particle bombardment SV40 lacZ [45]
Porphyra haitanensis stable glass bead agitation SV40 lacZ
EGFP		 [131]
Gracilaria changii stable particle bombardment SV40 lacZ [91]
Gracilaria gracilis stable particle bombardment SV40 lacZ [92]

Table 1.

Transformation in red seaweeds.

*Porphyra species used are P. yezoensis, P. tenera, P. okamurae, P. onoi, P. variegate and P. pseudolinearis.


2.2.1. Optimization of codon usage in the reporter gene

Inefficient expression of foreign genes in the green alga Chlamydomonas reinhardtii is often due to the incompatibility of the codon usage in the gene’s coding regions [49-51]. Expressed sequence tag (EST) analysis of P. yezoensis reveals that the codons in P. yezoensis nuclear genes frequently contain G and C residues especially in their third letters, by which means the GC content reaches a high of 65.2% [52]. Since bacterial GUS and lacZ reporter genes have AT-rich codons, the incompatibility of codon usage, which generally inhibits the effective use of transfer RNA by rarely used codons in the host cells, thus decreasing the efficiency of the translation [53], might be responsible for the poor translation efficiency of foreign genes in P. yezoensis cells. It is therefore possible that modification of codon usage in the GUS gene would enable the efficient expression of this gene in P. yezoensis cells.

Accordingly, the codon usage of the GUS reporter gene was adjusted to that in the nuclear genes of P. yezoensis by introducing silent mutations [48], by which unfavorable or rare codons in the GUS reporter gene were exchanged for favorable ones without affecting amino acid sequences. The resultant artificially codon-optimized GUS gene was designated PyGUS, and its GC content was increased from 52.3% to 66.6% [48]. When the PyGUS gene directed by the CaMV 35S promoter was introduced into P. yezoensis gametophytic cells by particle bombardment, low but significant expression of the PyGUS gene was observed by histochemical detection and GUS activity test, indicating enhancement of the expression level of the GUS reporter gene [29,30,48]. Optimization of the codon usage of the reporter gene is therefore one of the important factors for successful expression in P. yezoensis cells [29,30,48].

2.2.2. Employment of endogenous strong promoters

The CaMV 35S promoter has very low activity in cells of green microalgae such as Dunaliella salina [54], Chlorella kessleri [55] and Chlorella vulgaris [56] and no activity in C. reinhardtii cells [57-59]. Thus, a low level of PyGUS expression under the direction of the CaMV 35S promoter is likely to be caused by the low activity of this promoter in P. yezoensis cells. A hint to overcoming this problem was that employment of strong endogenous promoters such as the β-Tub, RbcS2 and Hsp70 promoters results in the efficient expression of foreign genes in microalgae [60-65]. Therefore, it is likely that efficient expression of the PyGUS reporter gene in P. yezoensis cells is caused by the recruitment of endogenous strong promoters.

By comparison with steady-state expression levels by reverse transcription-polymerase chain reaction (PCR), we found two genes strongly expressed in P. yezoensis: genes encoding glyceraldehyde-3-phosphate dehydrogenase (PyGAPDH) and actin 1 (PyAct1) [29]. When the PyGUS gene fused with the 5’ upstream regions of these genes were introduced into gametophytic cells by particle bombardment, cells expressing the reporter gene and GUS enzymatic activity were dramatically increased [48,66]. These results indicate that employment of endogenous strong promoters is another important factor necessary for high-level expression of the reporter gene in P. yezoensis cells. In addition, the original GUS gene was not activated by PyGAPDH or PyAct1 promoter [29,30,48], demonstrating that the PyGUS gene and endogenous strong promoter have a synergistic effect on the efficiency of the expression in P. yezoensis cells (Figure 1A). Therefore, the combination of endogenous strong promoters with codon optimized reporter genes is critical for successful transient transformation in Porphyra species [29,30]. The established procedure of transient transformation is schematically represented in Figure 2.

2.2.3. Application of the transient transformation for using fluorescent proteins

The GUS reporter gene is usually used to monitor gene expression in planta; however, visualization of the reporter products requires cell killing. Reporters that function in living cells have also been established to date with fluorescent proteins used most commonly. The green fluorescent protein (GFP) has the advantage over other reporters for monitoring subcellular localization of proteins in living cells, because its fluorescence can be visualized without additional substrates or cofactors [67]. At present, there are GFP variants with non-overlapping emission spectra such as cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein, which allows multicolor imaging in cells [68,69].

Until recently, there was no report about the successful expression of fluorescent proteins in seaweeds. However, based on an efficient transient transformation system in P. yezoensis, fluorescent reporter systems have recently been established in P. yezoensis [29,30,70,71]. The humanized fluorescent protein genes, AmCFP, ZsGFP, and ZsYFP (Clontech) and the plant-adapted GFP(S65T) [72], the GC contents of which are as high as 63.7%, 62.8%, 61.9% and 61.4%, respectively, were strongly expressed in gametophytic cells under the direction of the PyAct1 promoter using the particle bombardment method [71] (see Figure 1B).

The analysis of subcellular localization of cellular molecules was available using humanized and plant-adapted fluorescent reporters. The first successful attempt at achieving this process was to monitor the plasma membrane localization of phosphoinositides in P. yezoensis [70]. Phosphoinositides (PIs), whose inositol ring has hydroxyl groups at positions D3, D4 and D5 for phosphorylation, constitute a family of structurally related lipids, PtdIns-monophosphates [PtdIns3P, PtdIns4P and PtdIns5P], PtdIns-bisphosphates [PtdIns(3,4)P2, PtdIns(3,5)P2 and PtdIns(4,5)P2] and PtdIns-trisphosphate [PtdIns(3,4,5)P3], all of which are detectable in plants except for PtdIns(3,4,5)P3 [73,74]. Although the PIs are a minority among membrane phospholipids, they play important roles in regulating multiple processes of development and cell responses to environmental stimuli in land plants and green algae [74,75]. Recently, Li et al. [76,77] demonstrated that PIs are involved in the establishment of cell polarity in P. yezoensis monospores. The Pleckstrin homology (PH) domain, a PI-binding module, each part of which has individual substrate specificity, is usually used to monitor PIs in vivo by fusion with a fluorescent protein [78-80]. For instance, the PH domains from human phospholipase Cδ1 (PLCδ1) are employed for the detection of PtdIns(4,5)P2 [81], whereas that from the v-akt murine thymoma viral oncogene homolog 1 (Akt1) has dual specificity in the detection of both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 [82]. Because of this substrate specificity, we were able to visualize PtdIns(3,4)P2 and PtdIns(4,5)P2 at the plasma membrane with humanized AmCFP and ZsGFP fused to the PH domains from PLCδ1 and Akt1 via the direction of the PyAct1 promoter [70].

Figure 1.

Efficient expression of PyGUS and fluorescent proteins by the transeint transformation with circular expression plasmids in P. yezoensis gametophytic cells. (A) Expression of the codon-optimized PyGUS reporter gene under the direction of the actin 1 (PyAct1) promoter. Blue histochemically stained cells are PyGUS expression cells. Scale bar corresponds to 100 μm. (B) Expression of humanized AmCFP and plant-adapted sGFP(S65T). Gametophytic cells transiently transformed with expression plasminds containng AmCFP or sGFP(S65T) gene under the control of the PyAct1 promoter. Left and right panels show bright field and fluorescence images, respectively. Scale bar corresponds to 5 μm.

Figure 2.

The established procedure of transeient transformation in P. yezoensis. A circular expression plasmid is bombarded into P. yezoensis gametophytic cells using the Bio-Rad PDS-1000/He after coating of gold particles with the plasmid. Expression of the reporter gene is observed after cultivation of the bombareded gametophyte under dark for two days; for PyGUS reporter gene, histochemical staining with X-gluc solution and fluorometric analysis of enzymatic activity are performed; for fluorescent reporter genes, bombarded sanples are examined with fluorescent microscopy.

Moreover, subcellular localization of transcription factors was also visualized in P. yezoensis. When complete open reading frames (ORFs) of transcription elongation factor 1 (PyElf1) and multiprotein bridging factor 1 (PyMBF1) from P. yezoensis were fused to AmCFP or ZsGFP, nuclear localization of these fusion proteins was observed in gametophytic cells, which was confirmed by overlapping of fluorescent signals with SYBR Gold staining of the nucleus [71]

With the successfull visualization of subcellular localization of cellular molecules, the transient transformation system developed in P. yezoensis appearst to be powerful tool to analyze functions of genes and cellular components [29,30].

2.2.4. Applicability of the P. yezoensis transient transformation system in other red seaweeds

As described above, both the adjustment of codon usage of the reporter gene according to algal preference and the employment of the strong endogenous promoters are important for providing highly efficient and reproducible expression of the reporter gene in P. yezoensis. In addition to Bangiophyceae like Porphyra species, Florideophyceae are also known, including a number of industrially important species such as Gracilaria and Gelidium as sources of agar and Chondrus and Kappaphycus as sources of carrageenan. Thus, the establishment of a genetic manipulation system for both Bangiophyceae and Florideophyceae other than P. yezoensis is awaited. EST analysis of P. haitanensis revealed that the GC content of the ORFs in this alga was as high as that in P. yezoensis, and analysis of the GAPDH gene from a Florideophycean alga Chondrus crispus showed a high GC content (approximately 60%) in the coding region [83,84], which is consistent with the codon preference in P. yezoensis. Since efficient expression of the GAPDH-PyGUS gene has recently been confirmed in P. tenera [85], the applicability of the P. yezoensis transient gene expression system in other red seaweeds is expected. Indeed, using the PyGUS and sGFP(S65T) reporter genes under the direction of the PyAct1 promoter, efficient expression of PyGUS and sGFP(S65T) genes was observed in Bangiophyceae including P. tenera, P. okamurae, P. psedolinearis and Bangia fuscopurpurea, although the expression efficiency varied among species [86]. Thus, the transient transformation system developed in P. yezoensis is widely applicable in Bangiophycean red algae [29,30,86].

No expression of the reporter genes was seen in Florideophyceae [29,30,86]. Since the availability of the P. yezoensis promoter is responsible for this deficiency in gene expression, it is important to employ the 5’ upstream region of the suitable endogenous gene from Florideophycean algae. Alternatively, it is possible that the efficiency of plasmid transfer by bombardment parameters is reduced by the cell wall and thus the size of the gold particles, target distance, acceleration pressure and/or amount of DNA per bombardment should be adjusted.

Taken together, PyGUS and sGFP(S65T) genes act synergistically with the PyAct1 promoter as a heterologous promoter for transient transformation in Bangiophycean algae. Recently, the same synergistic effect was found in P. tenera; that is, Son et al. [85] clearly indicated that the heat shock protein 70 (PtHSP70) promoter from P. tenera can activate the PyGUS gene in gametophytic cells of this alga. Moreover, the PtHSP70-PyGUS gene was expressed in P. yezoensis, P. okamurae, P. psedolinearis and B. fuscopurpurea [85,87]. These findings are consistent with the importance of two critical factors for transient transformation in red seaweeds, adjustment of the codon usage in reporter genes and employment of a strong endogenous promoter.

The other important message gleaned from this experimental data is the efficient heterologous activation of PyGAPDH and PtHSP70 promoters in P. tenera and P. yezoensis, respectively [85,87]. For the genetic transformation, the target site for recombination is usually determined by the DNA sequence of genes desired for disruption or modification. Thus, it is better to exclude a possibility of homologous recombination at the DNA region corresponding to the promoter sequence used for expression of the reporter gene that is usually sandwiched between two different DNA sequences from the objective gene or its flanking regions. To avoid incorrect recombination at the promoter region, it is critical to employ heterologous promoters, whose sequence has low homology to the genome sequence of the host, to direct the expression of reporter genes. It is therefore possible that PyGAPDH and PtHSP70 promoters are useful for genetic transformation in P. tenera and P. yezoensis, respectively. The number of promoters acting for heterologous reporter gene expression in red algae must be increased to develop a sophisticated system for red algal genetic transformation.

2.3. Towards genetic transformation in red seaweeds

The successful genetic transformation in red alga has been established only in unicellular algae [20,88]. The first report described chloroplast transformation in the unicellular red alga Porphyridium sp. through integration of the gene encoding AHAS(W492S) into the chloroplast genome by homologous recombination, resulting in sulfometuron methyl (SMM) resistance at a high frequency in SMM-resistant colonies [89]. The next report was of stable nuclear transformation in the unicellular red alga Cyanidioschyzon merolae, for which the uracil auxotrophic mutant lacking the URA5.3 gene was used for the genetic background to isolate mutants with uracil prototrophic by employing the wild-type URA5.3 gene fragment as a selection maker [90].

Table 1 shows preliminary experiments with red seaweeds. The first was by Cheney et al. [26], who introduced the CaMV 35S-GUS and CaMV 35S-GFP genes in P. yezoensis genome via an Agrobacterium-mediated transformation system. In addition, they transformed P. yezoensis with a bacterial nitroreductase gene via an Agrobacterium-mediated method [28] and P. leucosticte monospores with an unknown gene by electroporation [27]. However, these reports appeared on conference abstracts and thus details of experimental procedures are unknown. In related work, the genetic transformation of Gracilaria species was recently reported [91,92], in which integration of the SV40-lacZ gene was checked by PCR using genomic DNAs prepared from particle-bombarded seaweeds; however, selection of transformed cells was not performed. Taken together, these preliminary experiments are not enough to conclude the establishment of genetic transformation in red seaweeds, meaning that the genetic transformation system has not yet been fully established in red macroalgae.

As mentioned above, procedures of integration and targeting of foreign genes into the genome and selection of transformed cells must be developed for establishing the genetic transformation system, although other requirements such as an efficient gene transfer system and an efficient expression system for foreign genes have been resolved by developing the transient transformation system in Bangiophyceae [29,30]. Regarding the unresolved points, knowledge about the selection of transformed cells is now accumulating. Selection marker genes are required to distinguish between transformed cells and non-transformed cells, since successful integration of a foreign gene into the host genome usually occur in only a small percentage of transfected cells. These genes confer new traits to any transformed target strain of a certain species, thus enabling the transformed cells to survive on medium containing the selective agent, where non-transformed cells die. Genes with resistance to the aminoglycoside antibiotics, which bind to ribosomal subunits and inhibit protein synthesis in bacteria, eukaryotic plastids and mitochondria [93], are generally used as selection markers. For example, the antibiotics hygromycin and geneticin (G418) are frequently used as selection agents with the hygromycin phosphotransferase (hptII) gene to inactivate hygromycin via an ATP-dependent phosphorylation [94] and the neomycin phosphotransferase II (nptII) gene to detoxify neomycin, G418 and paromomycin [93], respectively. In the green alga Chlamydomonas reinhardtii, the hygromycin phosphotransferase (aph7”) gene from Streptomyces hygroscopicus and the aminoglycoside phosphotransferase aphVIII (aphH) gene from S. rimosus had been reported as selectable marker genes for hygromycin and paromomycin, respectively, with similarity in the codon usage [95-97]. The aphH gene from S. rimosus is also applicable to the multicellular green alga Volvox carteri as a paromomycin-resistance gene [97,98]. In the diatom Phaeodactylum tricornutum, the expressed chloramphenicol acetyltransferase gene (CAT) detoxifies chloramphenicol [99], and the nptII gene confers resistance to the aminoglycoside antibiotic G418 [64]. Likewise, the nptII gene gives resistance to the antibiotic G418 in the diatoms Navicula saprophila and Cyclotella cryptica [100]. However, it is unknown what kinds of antibiotics-based selection marker genes are available for red seaweeds, since red algae usually have strong resistance to antibiotics.

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. According to these findings, it is necessary to confirm whether P. yezoensis gametophytes will obtain antibiotic tolerance by introducing plasmid constructs containing the antibiotic-resistance genes mentioned above. In this case, optimization of codon usage and the employment of strong endogenous promoter are expected for functional expression of the antibiotic resistance genes, according to the knowledge from the transient transformation system [29,30]. Such efforts could effectively contribute to the establishment of the genetic transformation system in red seaweeds in the near future.

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3. Transformation in brown seaweeds

According to Qin et al. [103], trials of genetic engineering in brown seaweeds have been started by transient expression of the GUS reporter gene under direction of the CaMV 35S promoter by particle bombardment in Laminaria japonica and Undaria pinnatifida, which were first performed in 1994 by them. Descriptions of related experiments were published later [104,105]. Qin et al. then focused on the establishment of genetic transformation in brown seaweeds and provided successful reports of genetic transformation in L. japonica [103,106]. Genetic transformation was performed by particle bombardment only and expression of a reporter gene was driven by the SV40 promoter that is usually used for gene expression in mammalian cells (Table 2). This promoter represented non-tissue and -cell specificity for expression of the E. coli lacZ reporter gene [105]. Promoters from maize ubiquitin, algal adenine-methyl transfer enzyme and diatom fucoxanthin chlorophyll a/c-binding protein (FCP) genes are also useful for transient expression of the GUS gene, and the FCP promoter is also employable for the genetic transformation [107]. Interestingly, there has been no successful genetic transformation using the CaMV 35S promoter, although this promoter is active in the transient transformation [103].

Despite the reports of successful genetic transformation, there was no experiment using antibiotics-based selection of transformants in brown seaweeds. Although the susceptibility of brown seaweeds to antibiotics has not been well studied, it was reported that L. japonica was sensitive to chloramphenicol and hygromycin, but not to ampicillin, streptomycin, kanamycin, neomycin or G418 [103,106]. Since hygromycin is more effective than chloramphenicol [103,106], it is necessary to confirm the utility of the SV40-hptII gene for the selection of transformants to fully establish the genetic transformation system in kelp.

Species Status of expression Gene transfer method Promoter Marker or Reporter Ref.
Laminaria japonica transient particle bombardment CaMV 35S GUS [103]
Laminaria japonica stable particle bombardment SV40 GUS [105]
Laminaria japonica transient particle bombardment CaMV 35S, UBI, AMT GUS [107]
Laminaria japonica stable particle bombardment FCP GUS [107]
Laminaria japonica stable particle bombardment SV40 HBsAg [113]
Laminaria japonica stable particle bombardment SV40 Rt-PA [114]
Laminaria japonica stable particle bombardment SV40 bar [114]
Undaria pinnatifida transient particle bombardment CaMV 35S GUS [103]
Undaria pinnatifida transient particle bombardment SV40 GUS [104]

Table 2.

Transformation in brown seaweeds.

To date, stably transformed microalgae have been employed to produce recombinant antibodies, vaccines or bio-hydrogen as well as to analyze the gene functions targeted for engineering [108-111]. Based on the success in genetic transformation, L. japonica is now proposed as a marine bioreactor in combination with the SV40 promoter [112]. Indeed, the integration of human hepatitis B surface antigen (HBsAg) and recombinant human tissue-type plasminogen (rt-PA) genes into the L. japonica genome resulted in the efficient expression of these genes under the direction of the SV40 promoter [113,114]. Therefore, L. japonica promises to be useful as the bioreactor for vaccine and other medical agents, although it is necessary to continually check the safety and value of its use by oral application.

There is no competitor against the Chinease group in the field of using brown algal genetic transformation at present [103,106,115], meaning there is currently no way to confirm the replicability of the experiments. It is necessary to re-examine the effective use of the non-plant SV40 promoter and bacterial lacZ gene in brown algal genetic transformation, which is also important for the evaluation of genetic transformation in red seaweeds Gracilaria species, for which the SV40-lacZ gene was used such as transgene, as described above [91,92].

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4. Transformation in green seaweeds

The first successfull genetic transformation in green algae was reported in the unicellular green alga Chlamydomonas reinhardtii for which the particle bombardment and glass-bead abrasion techniques were employed [116,117]. The availability of electoroporation was then confirmed in C. reinhardtii and Chlorella saccharophila [118,119]. These methods produce physical cellular damage, allowing DNA to be introduced into the cells. Moreover, particle bombardment was confirmed to be useful for a diverse range of species, including transient transformation in the unicellular Haematococcus pluvialis [120] and genetic transformation in the multicellular Volvox carteri and Gonium pectoral [97,120-122]. Agrobacterium-mediated transformation was also reported in H. pluvialis [123]. Thus, all methods employed in land green plants are applicable for green microalgae [88] (see Table 3).

In contrast, there is no report about genetic transformation in green seaweeds (Table 3). To date, only two examples of transient transformation have been reported in green seaweeds, Ulva lactura by electroporation and U. pertusa by particle bombardment [124,125]. As shown in Table 3, some of the experiments with micro- and macro-green algae used the promoter of the CaMV 35S gene and the coding region of the E. coli GUS gene. Although functionality of the CaMV 35S promoter and bacterial GUS coding region is the same in land green plants, the expression of the GUS reporter gene seems to be very low in the green seaweed U. lactuca [124]. In fact, codon-optimization is critical for the expression of reporters like the GFP gene and antibiotic-resistance genes in C. reinhardtii [47,90,115,126]. Moreover, the HSP70A promoter was employed to increase the expression level of the reporter genes [47,115]. Therefore, it is possible that changes in codon usage in the reporter gene and promoter region could result in increased reporter gene expression in transient transformation of green seaweeds. Recently, the Rubisco small subunit (rbsS) promoter was used for expression of the EGFP reporter gene in transient transformation of U. pertusa by particle bombardment [125]; however, it is still unclear whether the rbsS promoters and the EGFP gene work well in cells in comparison with the CaMV 35S promoter and codon-optimized EGFP gene.

Species Status of expression Gene transfer method Promoter Marker or Reporter Ref.
Microalga
Chlamidominas reinhardtii stable particle bombardment [116]
Chlamidominas reinhardtii stable glass bead agitation Nitrate reductase Nitrate reductase [117]
Chlamidominas reinhardtii stable electroporation CaMV 35S CAT [118]
Chlamidominas reinhardtii stable glass bead agitation rbcS2 aphVIII [95]
Chlamidominas reinhardtii stable glass bead agitation β2-tubulin Aph7” [96]
Chlorella saccharophila transient electroporation CaMV 35S GUS [119]
Haematococcus pluvialis transient particle bombardment SV40 lacZ [120]
Haematococcus pluvialis stable Agrobacterium-mediated
gene transfer		 CaMV 35S GUS,GFP,
hptII		 [123]
Volvox Carteri stable particle bombardment β2-tubulin arylsulfatase [121]
Volvox Carteri stable particle bombardment
glass bead agitation		 Hsp70A-rbcS2 fusion aphVIII [98]
Volvox Carteri stable particle bombardment β-tubulin, Hsp70A aphH [97]
Gonium pectoral stable particle bombardment VcHsp70A aphVIII [122]
Seaweed
Ulva lactuca transient electroporation CaMV 35S GUS [124]
Ulva pertusa transient particle bombardment UprbcS EGFP [125]

Table 3.

Transformation in green algae.

If the rbsS-EGFP gene is useful as a reporter gene for genetic transformation in green seaweeds, the remaining problems to be settled are methods for foreign gene integration into the genome and selection of transformed cells, which is the same as the situation with red seaweeds. Reddy et al. [24] commented on the antibiotic sensitivity of green seaweeds, indicating the considerable resistance of protoplast from Ulva and Monostroma to hygromycin and kanamycin. Insensitivity to hygromycin is inconsistent with the case for red and brown seaweeds [101-103,106]. 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.

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5. Conclusion

It is nearly 20 years since the first transient transformation of a red seaweed with a circular expression plasmid [25], and many efforts have been made to develop a system for transient and stable expression of foreign genes in many kinds of seaweeds; however, a seaweed transformation system has still not been developed. The main problem is the employment of the CaMV 35S-GUS gene in the pioneer attempts at system development as shown in Tables 1, 2 and 3. This problem was recently resolved through the development of an efficient transient transformation system in P. yezoensis [29,30]. It is clear that the CaMV 35S promoter and the GUS gene are not active in seaweed cells [48], which is supported by knowledge from green microalgae [54-65]. These findings strongly indicate that defects in the transfer and expression of foreign genes were resolved by knowledge about two critical factors required for reproducibility and efficiency of transient gene expression, namely, the optimization of codon usage of coding regions and the employment of endogenous strong promoters [29,30]. However, these significant improvements are not enough to allow the establishment of a genetic transformation system in seaweeds.

At present, genetic transformation is reported in red and brown seaweeds using the SV40 promoter (Tables 1 and 2) [91,92,103,105-107,113,114]; however, isolation of transgenic clone lines produced from distinct single transformed cells, which is the final goal of the genetic transformation of seaweeds as a tool, has not been reported, and seaweed genetic transformation is thus not fully developed. 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 [101-103,106], 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. To this end, we must reevaluate the availability of the methods for gene transfer such as electroporation and Agrobacteriumu infection.

Due to the problems with efficient genetic transformation systems, the molecular biological studies of seaweeds are currently progressing more slowly than are the studies of land green plants. Since a genetic transformation system would allow us to perform genetic analysis of gene function via inactivation and knock-down of gene expression by RNAi and antisense RNA supression, its establishment will enhance both our biological understanding and genetical engineering for the sustainable production of seaweeds and also for the use of seaweeds as bioreactors.

References

  1. 1. Griffith, F. The significance of pneumococcal types. Journal of Hygiene 1928;27(2) 113–159.
  2. 2. Avery OT, MacLeod CM, MaCarty M. Studies on the chemical nature of the substance inducing transformation of Pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus Type III. Journal of Experimental Medicine 1944;79(2) 137–158.
  3. 3. Mandel M, Higa A. Calcium-dependent bacteriophage DNA infection. Journal of Molecular Biology 1970;53(1) 159–162.
  4. 4. Griesbeck C, Kobl I, Heitzer M. Chlamydomonas reinhardtii. A protein expression system for pharmaceutical and biotechnological proteins. Molecular Biotechnology 2006;34(2) 213-223.
  5. 5. Torney F, Moeller L, Scarpa A, Wang K. Genetic engineering approaches to improve bioethanol production from maize. Current Opinion in Biotechnology 2007;18(3) 193-199.
  6. 6. Bhatnagar-Mathur P, Vadez V, Sharma KK. Transgenic approaches for abiotic stress tolerance in plants: retrospect and prospects. Plant Cell Reports 2008;27(3) 411-424.
  7. 7. Doehmer J, Barinaga M, Vale W, Rosenfeld MG, Verma IM, Evans RM. Introduction of rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector: expression and regulation. Proceedings of the National Academy of Sciences of the United States of America 1982;79(7) 2268-7222.
  8. 8. Smith EF, Townsend CO. A plant tumor of bacterial origin. Science 1907;25(643) 671-673.
  9. 9. Chilton MD, Drummond MH, Merio DJ, Sciaky D, Montoya AL, Gordon MP, Nester EW. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 1977;11(2) 263-271.
  10. 10. Zambryski P, Joos H, Genetello C, Leemans J, Van Montagu M, Schell J. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO Journal 1983;2(12) 2143–2150.
  11. 11. Bevan M. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 1984;12(22) 8711-8721.
  12. 12. Guo M, Bian X, Wu X, Wu M. Agrobacterium-mediated genetic transformation: history and progress. In: Alvarrez MA (ed.) Genetic Transformation. Rijeka: InTech; 2011. p5-28.
  13. 13. Klein TM, Wolf ED, Wu R, Sanford JC. High-velocity microprojectiles for delivery of nucleic acids into living cells. Nature 1987;327(6117) 70-73.
  14. 14. Fromm ME, Taylor LP, Walbot V. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proceedings of the National Academy of Sciences of the United States of America 1985;82(17) 5824-5828.
  15. 15. Newell CA. Plant transformation technology: developments and applications. Molecular Biotechnology 2000;16(1) 53-65.
  16. 16. Radchuk VV, Ryschka U, Schumann G, Klocke E. Genetic transformation of cauliflower (Brassica oleracea var. botrytis) by direct DNA uptake into mesophyll protoplasts. Physiologia Plantarum 2002;114(3) 429-438.
  17. 17. Cove D. The moss Physcomitrella patens. Annual Review of Genetics 2005;39 339-358.
  18. 18. Takenaka M, Yamaoka S, Hanajiri T, Shimizu-Ueda Y, Yamato KT, Fukuzawa H, Ohyama K. Direct transformation and plant regeneration of the haploid liverwort Marchantia polymorpha L. Transgenic Research 2000;9(3) 179-185.
  19. 19. D’Orazio N, Gemello E, Bammoue MA, de Girolamo M, Ficoneri C, Riccioni G.: A treasure from the sea. Marine Drugs 2012;10(3) 604-616.
  20. 20. Hallmann A. Algal transgenics and biotechnology. Transgenic Plant Journal 2007;1(1) 81-98.
  21. 21. Smit AJ. Medicinal and pharmaceutical uses of seaweed natural products: a review. Journal of Applied Phycology 2004;16(4) 245-262.
  22. 22. van Ginneken VJTH, Helsper JPEG, de Visser W, van Keulen H, Brandenburg WA. Polyunsaturated fatty acids in various macroalgal species from north Atlantic and tropical seas. Lipids in Health and Disease 2011;10 104. (doi:10.1186/1476-511X-10-104) http://www.lipidworld.com/content/10/1/104 (accessed 22 June 2011).
  23. 23. Walker TL, Collet C, Purton S. Algal transgenics in the genomic era. Journal of Phycology 2005;41(6) 1077-1093.
  24. 24. Reddy CRK, Gupta MK, Mantri VA, Jha B. Seaweed protoplasts: status, biotechnological perspectives and needs. Journal of Applied Phycology 2008;20(5) 619-632.
  25. 25. Kurtzman AM, Cheney DP. Direct gene transfer and transient expression in a marine red alga using the biolistic method. Journal of Phycology 1991;27(Supplement) 42.
  26. 26. Cheney DP, Metz B, Stiller J. Agrobacterium-mediated genetic transformation in the macroscopic marine red alga Porphyra yezoensis. Journal of Phycology 2001;37(Supplement) 11–12.
  27. 27. Lin CM, Larsen J, Yarish C, Chen T. A novel gene transfer in Porphyra. Journal of Phycology 2001;37(Supplement) 31.
  28. 28. Bernasconi P, Cruz-Uribe T, Rorrer G, Bruce N, Cheney DP. Development of a TNT-detoxifying strain of the seaweed Porphyra yezoensis through genetic engineering. Journal of Phycology 2004;40(Supplement) 31.
  29. 29. Mikami K, Hirata R, Takahashi M, Uji T, Saga N. Transient transformation of red algal cells: Breakthrough toward genetic transformation of marine crop Porphyra species. In: Alvarez MA. (ed.) Genetic Transformation. Rijeka: InTech; 2011. p241-258.
  30. 30. Mikami K, Uji T. Transient gene expression systems in Porphyra yezoensis: Establishment, application and limitation. In: Mikami K. (ed.) Porphyra yezoensis: Frontiers in Physiological and Molecular Biological Research. New York: Nova Science Publishers; 2012. p93-117.
  31. 31. Basu C, Kausch AP, Chandlee JM. Use of β-glucuronidase reporter gene for gene expression analysis in turfgrasses. Biochemical and Biophysical Research Communications 2004;320(1) 7-10.
  32. 32. Sun P, Tian QY, Chen J, Zhang WH. Aluminium-induced inhibition of root elongation in Arabidopsis is mediated by ethylene and auxin. Journal of Experimantal Botany 2010;61(2) 347-356.
  33. 33. Jefferson RA. The GUS reporter gene system. Nature 1989;342(6251) 837-838.
  34. 34. Cervera M. Histochemical and fluorometric assays for uidA (GUS) gene detection. Methods in Moecular Biology 2004;286(4) 203-213.
  35. 35. Louis J, Lorenc-Kukula K, Singh V, Reese J, Jander G, Shah J. Antibiosis against the green peach aphid requires the Arabidopsis thaliana MYZUS PERSICAEINDUCED LIPASE1 gene. Plant Journal 2010;64(5) 800-811.
  36. 36. Wally O, Punja ZK. Enhanced disease resistance in transgenic carrot (Daucus carota L.) plants over-expressing a rice cationic peroxidase. Planta 2010;232(5) 1229-1239.
  37. 37. Kübler JE, Minocha SC, Mathieson AC. Transient expression of the GUS reporter gene in protoplasts of Porphyra miniata (Rhodophyta). Journal of Marine Biotechnology 1994;1 165–169.
  38. 38. Kuang M, Wang SJ, Li Y, Shen DL, Zeng CK. Transient expression of exogenous GUS gene in Porphyra yezoensis (Rhodophyta). Chinese Journal of Oceanology and Limnology 1998;16(1) 56–61.
  39. 39. Okauchi M, Mizukami Y. Transient β-Glucuronidase (GUS) gene expression under control of CaMV 35S promoter in Porphyra tenera (Rhodophyta). Bulletin of National Research Institute of Aquaculture 1999;Supplement 4 13-18.
  40. 40. Hado M, Okauchhi M, Murase N, Mizukami Y. Transient expression of GUS gene using Rubisco gene promoter in the protoplasts of Porphyra yezoensis. Suisan Zoushoku 2003;51(3) 355-360.
  41. 41. Liu HQ, Yu WG, Dai JX, Gong QH, Yang KF, Zhang YP. Increasing the transient expression of GUS gene in Porphyra yezoensis by 18S rDNA targeted homologous recombination. Journal of Applied Phycology 2003;15(5) 371-377.
  42. 42. Gong Q, Yu W, Dai J, Liu H, Xu R, Guan H, Pan K. Efficient gusA transient expression in Porphyra yezoensis protoplasts mediated by endogenous beta-tubulin flanking sequences. Journal of Ocean University of China 2005;6(1) 21-25.
  43. 43. Bell P, Limberis M, Gao GP, Wu D, Bove MS, Sanmiguel JC, Wilson JM. An optimized protocol for detection of E. coli beta-galactosidase in lung tissue following gene transfer. Histochemistry and Cell Biology 2005;124(1) 77-85.
  44. 44. Gan SY, Qin S, Othman RY, Yu D, Phang SM. Transient expression of lacZ in particle bombarded Gracilaria changii (Gracilariales, Rhodophyta). Journal of Applied Phycology 2003;15(4) 351–353.
  45. 45. Wang J, Jiang P, Cui Y, Deng X, Li F, Liu J, Qin S. Genetic transformation in Kappaphycus alvarezii using micro-particle bombardment: a potential strategy for germplasm improvement. Aquaculture International 2010;18(6) 1027-1034.
  46. 46. Kang HG, An GH. Morphological alterations by ectopic expression of the rice OsMADS4 gene in tobacco plants. Plant Cell Reports 2005;24(2) 120-126.
  47. 47. Funabashi H, Takatsu M, Saito M, Matsuoka H. Sox2 regulatory region 2 sequence works as a DNA nuclear targeting sequence enhancing the efficiency of an exogenous gene expression in ES cells. Biochemical and Biophysical Research Communications 2010;400(4) 554-558.
  48. 48. Fukuda S, Mikami K, Uji T, Park EJ, Ohba T, Asada K, Kitade Y, Endo H, Kato I, Saga N. Factors influencing efficiency of transient gene expression in the red macrophyte Porphyra yezoensis. Plant Science 2008;174(3) 329-339.
  49. 49. Fuhrmann M, Hausherr A, Ferbitz L, Schödl T, Heitzer M, Hegemann P. Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Molecular Biology 2004;55(6) 869-881.
  50. 50. Ruecker O, Zillner K, Groebner-Ferreira R, Heitzer M. Gaussia-luciferase as asensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii. Molecular Genetics and Genomics 2008;280(2) 153-162.
  51. 51. Shao N, Bock R. A codon-optimized luciferase from Gaussia princepsfacilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii. Current Genetics 2008;53(6) 381-388.
  52. 52. Nikaido I, Asamizu E, Nakajima M, Nakamura Y, Saga N, Tabata S. Generation of 10,154 expressed sequence tags from a leafy gametophyte of a marine red alga, Porphyra yezoensis. DNA Research 2000;7(3) 223-227.
  53. 53. Mayfield SP, Kindle KL. Stable nuclear transformation of Chlamydomonas reinhardtii by using a C. reinhardtii gene as the selectable marker. Proceedings of the National Academy of Sciences of the United States of America 1990;87(6) 2087-2091.
  54. 54. Tan D, Qin S, Zhang Q, Jiang P, Zhao F. Establishment of a micro-particle bombardment transformation system for Dunaliella salina. Journal of Microbiology 2005;43(4) 361-365.
  55. 55. El-Sheekh MM. Stable transformation of the intact cells of Chlorella kessleri with high velocity microprojectiles. Biologia Plantarum 1999;42(2) 209-216.
  56. 56. Chow KC, Tung WL. Electrotransformation of Chlorella vulgaris. Plant Cell Reports 1999;18(9) 778-780.
  57. 57. Day A, Debuchy R, Dillewijn J, Purton S, Rochaix JD. Studies on the maintenance and expression of cloned DNA fragments in the nuclear genome of the green alga Chlamydomonas reinhardtii. Physiologia Plantarum 1990;78(2) 254-260.
  58. 58. Blankenship JE, Kindle K. Expression of chimeric genes by the light-regulated cabII-1 promoter in Chlamydomonas reinhardtii: a cabII-1/nit1 gene functions as a dominant selectable marker in a nit1- nit2-strain. Molecular and Cellular Biology 1992;12(11) 5268-5279.
  59. 59. Lumbreras V, Stevens DR, Purton S. Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant Journal 1998;14(4) 441-447.
  60. 60. Davies JP, Weeks DP, Grossman AR. Expression of the arylsulfatase gene from the beta 2-tubulin promoter in Chlamydomonas reinhardtii. Nucleic Acids Res 1992;20(12) 2959-2965.
  61. 61. Stevens DR, Rochaix JD, Purton S. The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Molecular and General Genetics 1996;251(1) 23-30.
  62. 62. Schroda M, Blocker D, Beck CF. The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant Journal 2000;21(2) 121-131.
  63. 63. Walker TL, Becker DK, Collet CC. Characterisation of the Dunaliella tertiolecta RbcS genes and their promoter activity in Chlamydomonas reinhardtii. Plant Cell Reports 2004;23(10-11) 727-735.
  64. 64. Zaslavskaia LA, Lippmeier JC, Kroth PG, Grossman AR, Apt KE. Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. Journanl of Phycology 2000;36(2) 379-386.
  65. 65. Hirakawa Y, Kofuji R, Ishida K. Transient transformation of achlorarachniophyte alga, Lotharella amoebiformis (Chlorarachniophyceae), with uidA and egfp reporter genes. Jounal of Phycology 2008;44(3) 814-820.
  66. 66. Takahashi M, Uji T, Saga N, Mikami K. Isolation and regeneration of transiently transformed protoplasts from gametophytic blades of the marine red alga Porphyra yezoensis. Electronic Journal of Biotechnology 2010;13(2) (doi:10.2225/vol13-issue2-fulltext-7) http://www.ejbiotechnology.cl/content/vol13/issue2/full/7/index.html (accessed 15 March 2010).
  67. 67. Ehrhardt D. GFP technology for live cell imaging. Current Opinion in Plant Biology 2003;6(6) 622-628.
  68. 68. Lin ZF, Arciga-Reyes L, Zhong SL, Alexander L, Hackett R, Wilson I, Grierson D. SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development. Journal of Experimental Botany 2008;59(15) 4271-4287.
  69. 69. Martin K, Kopperud K, Chakrabarty R, Banerjee R, Brooks R, Goodin MM. Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta. Plant Journal 2009;59(1) 150-162.
  70. 70. Mikami K, Uji T, Li L, Takahashi M, Yasui H, Saga N. Visualization of phosphoinositides via the development of the transient expression system of a cyan fluorescent protein in the red alga Porphyra yezoensis. Marine Biotechnology 2009;11(5) 563-569.
  71. 71. Uji T, Takahashi M, Saga N, Mikami K. Visualization of nuclear localization of transcription factors with cyan and green fluorescent proteins in the red alga Porphyra yezoensis. Marine Biotechnology 2010;12(2) 150-159.
  72. 72. Niwa Y, Hirano T, Yoshimoto K, Shimizu M, Kobayashi H. Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants. Plant Journal 1999;18(4) 455-463.
  73. 73. Xue HW, Chen X, Me Y. Function and regulation of phospholipid signaling in plants. Biochemical Journal 2009;421(Part 2) 145-156.
  74. 74. Heilmann I. Using genetic tools to understand plant phosphoinositide signalling. Trends in Plant Science 2009;14(3) 171-179.
  75. 75. Williams ME, Torabinejad J, Cohick E, Parker K, Drake EJ, Thompson JE, Hortter M, DeWald DB. Mutations in the Arabidopsis phosphoinositide phosphatase gene SAC9 lead to over accumulation of PtdIns(4,5)P2 and constitutive expression of the stress-response pathway. Plant Physiology 2005;138(2) 686-700.
  76. 76. Li L, Saga N, Mikami K. Phosphatidylinositol 3-kinase activity and asymmetrical accumulation of F-actin are necessary for establishment of cell polarity in the early development of monospores from the marine red alga Porphyra yezoensis. Journal of Experimental Botany 2008;59(13) 3575-3586.
  77. 77. Li L, Saga N, Mikami K. Ca2+ influx and phosphoinositide signalling are essential for the establishment and maintenance of cell polarity in monospores from the red alga Porphyra yezoensis. Journal of Experimental Botany 2009;60(12) 3477-3489.
  78. 78. Vermeer JEM, Thole JM, Goedhart J, Nielsen E, Munnik T, Gadella TW. Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells. Plant Journal 2009;57(2) 356-372.
  79. 79. Szentpetery Z, Balla A, Kim YJ, Lemmon MA, Balla T. Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study. BMC Cell Biology 2009;10 67. (doi:10.1186/1471-2121-10-67) http://www.biomedcentral.com/1471-2121/10/67 (accessed 21 September 2009).
  80. 80. Loovers HM, Postma M, Keizer-Gunnink I, Huang YE, Devreotes PN, van Haastert PJ. Distinct roles of PI(3,4,5)P3 during chemoattractant signaling in Dictyostelium: a quantitative in vivo analysis by inhibition of PI3-kinase. Molecular Biology of the Cell 2006;17(4) 1503-1513.
  81. 81. Lee Y, Kim YW, Jeon BW, Park KY, Suh SJ, Seo J, Kwak JM, Martinoia E, Hwang I. Phosphatidylinositol 4,5-bisphosphate is important for stomatal opening. Plant Journal 2007;52(5) 803-816.
  82. 82. Nishio M, Watanabe KI, Sasaki J, Taya C, Takasuga S, Iizuka R, Balla T, Yamazaki M, Watanabe H, Itoh R, Kuroda S, Horie Y, Forster I, Mak TW, Yonekawa H, Penninger JM, Kanaho Y, Suzuki A, Sasaki T. Control of cell polarity and motility by the PtdIns(3,4,5)P-3 phosphatase SHIP1. Nature Cell Biology 2007;9(1) 36-44.
  83. 83. Fan XL, Fang YJ, Hu SN, Wang GC. Generation and analysis of 5318 expressed sequence tags from the filamentous sporophyte of Porphyra haitanensis (Rhodophyta). Journal of Phycology 2007;43(6) 1287–1294.
  84. 84. Liaud MF, Valentin C, Brandt U, Bouget FY, Kloareg B, Cerff R. (1993). The GAPDH gene system of the red alga Chondrus crispus: promoter structures, intron/exon organization, genomic complexity and differential expression of genes. Plant Molecular Biology 1993;23(5) 981–994.
  85. 85. Son SH, Ahn J-W, Uji T, Choi D-W, Park E-J, Hwang MS, Liu JR, Choi D, Mikami K, Jeong W-J. Development of a transient gene expression system in the red macroalga, Porphyra tenera. Journal of Applied Phycology 2012;24(1) 79-87.
  86. 86. Hirata R, Takahashi M, Saga N, Mikami K. Transient gene expression system established in Porphyra yezoensis is widely applicable in Bangiophycean algae. Marine Biotechnology 2011;13(5) 1038-1047.
  87. 87. Hirata R, Jeong W-J, Saga N, Mikami K. Heterologous activation of the Porphyra tenera HSP70 promoter in Bangiophycean algal cells. Bioengineered Bugs 2011;2(5) 272-274.
  88. 88. Coll JM. Methodologies for transferring DNA into eukaryotic microalgae. Spanish Journal of Agricultural Research 2006;4(4) 316-330.
  89. 89. Lapidot M, Raveh D, Sivan A, Arad S, Shapira M. Stable Chloroplast transformation of the unicellular red alga Porphyridium species. Plant Physiology 2002;129(1) 7-12.
  90. 90. Minoda A, Sakagami R, Yagisawa F, Kuroiwa T, Tanaka K. Improvement of culture conditions and evidence for nuclear transformation by homologous recombination in a red alga, Cyanidioschyzon merolae 10D. Plant and Cell Physiology 2004;45(6) 667-671.
  91. 91. Gan SY, Qin S, Othman RY, Yu D, Phang SM. Development of a transformation system for Gracilaria changii (Gracilariales, Rhodophyta), a Malaysian red alga via microparticle bombardment. The 4 th Annual Seminar of National Science Fellowship 2004, 2004;BIO08, 45-48.
  92. 92. Haddy SM, Meyers AE, Coyne VE. Transformation of lacZ using different promoters in the commercially important red alga, Gracilaria gracilis. Afreican Journal of Biotechnology 2012;11(8) 1879-1885.
  93. 93. Miki B, McHugh S. Selectable marker genes in transgenic plants: applications, alternatives and biosafety. Journal of Biotechnology 2004;107(3) 193-232.
  94. 94. Tian LN, Charest PJ, Seguin A, Rutledge RG. Hygromycin resistance is an effective selectable marker for biolistic transformation of Black spruce (Picea mariana). Plant Cell Reports 2000;19(4) 358-362.
  95. 95. Sizova I, Fuhrmann M, Hegemann P. A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 2001;277(1-2) 221-229.
  96. 96. Berthold P, Schmitt R, Mages W. An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist 2002;153(4) 401-412.
  97. 97. Jakobiak T, Mages W, Scharf B, Babinger P, Stark K, Schmitt R. The bacterial paromomycin resistance gene, aphH, as a dominant selectable marker in Volvox carteri. Protest 2004;155(4) 381-393.
  98. 98. Hallmann A, Wodnniok S. Swapped green algal promoters: aphVIII-based gene constructs with Chlamydomonas flanking sequences work as dominant selectable makers in Volvox and vice versa. Plant Cell Reports 2006;25(6) 582-591.
  99. 99. Apt KE, Kroth-Pancic PG, Grossman AR. Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Molecular and General Genetics 1996;252(5) 572-579.
  100. 100. Dunahay TG, Jarvis EE, Roessler PG. Genetic transformation of the diatom Cyclotella cryptic and Navicula saprophila. Journal of Phycology 1995;31(6) 1004-1012.
  101. 101. Takahashi M, Mikami K, Mizuta H, Saga N. Identification and efficient utilization of antibiotics for the development of a stable transformation system in Porphyra yezoensis (Bangiales, Rhodophyta). Journal of Aquaculture Research and Development 2011;2 118, (doi:10.4172/2155-9546.1000118). http://www.omicsonline.org/2155-9546/2155-9546-2-118.php (accessed 23 December 2011)
  102. 102. Lee YK, An G, Lee IK. Antibiotics resistance of a red alga, Griffithsia japonica. Journal of Plant Biology 2000;43(2) 179-182.
  103. 103. Qin S, Jiang P, Li X, Wang X, Zeng C. A transformation model for Laminaria japonica (Phaeophyta, Laminariales). Chinese Journal of Oceanology and Limnology 1998;16(Supplement 1) 50-55.
  104. 104. Yu D, Qin S, Sun G, Chengkui Z. Transient expression of lacZ in the economic seaweed Undaria pinnatifida. High Technology Letters 2002;12(8) 93-95.
  105. 105. Jiang P, Qin S, Tseng CK. Expression of the lacZ reporter gene in sporophytes of the seaweed Laminaria japonica (Phaeophyceae) by gametophyte-targeted transformation. Plant Cell Reports 2003;21(12) 1211-1216.
  106. 106. Qin S, Sun GQ, Jiang P, Zou LH, Wu Y, Tseng C. Review of genetic engineering of Laminaria japonica (Laminariales, Phaepophyta) in China. Hydrobiologia 1999;398/399(0) 469-472.
  107. 107. Li F, Qin S, Jiang P, Wu Y, Zhang W. The integrative expression of GUS gene driven by FCP promoter in the seaweed Laminaria japonica (Phaeophyta). Journal of Applied Phycology 2009;21(3) 287-293.
  108. 108. Sun M, Qian KX, Su N, Chang HY, Liu JX, Chen GF. Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast. Biotechnology Letters 2003;25(13) 1087-1092.
  109. 109. Zorin B, Lu YH, Sizova I, Hegemann P. Nuclear gene targeting in Chlamydomonas as exemplified by disruption of the PHOT gene. Gene 2009;432(1-2) 91-96.
  110. 110. Specht E, Miyake-Stoner S, Mayfesqaxeld S. Micro-algae come of age as a platform for recombinant protein production. Biotechnology Letters 2010;32(10) 1373-1383.
  111. 111. Wu S, Huang R, Xu LL, Yan GY, Wang QX. Improved hydrogen production with expression of hemH and lba genes in chloroplast of Chlamydomonas reinhardtii. Journal of Biotechnology 2010;146(3) 120-125.
  112. 112. Qin S, Jiang P, Tseng CK. Transforming kelp into a marine bioreactor. Trends in Biotechnology 2005;23(5) 264-268.
  113. 113. Jiang P, Qin S, Tseng CK. Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta). Chinese Science Bulletin 2002;47(17) 1438-1440.
  114. 114. Zhang YC, Jiang P, Gao JT, Liao JM, Sun SJ, Shen ZL, Qin S. Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta). Science in China-Series C, Life sciences 2008;51(12) 1116-1120.
  115. 115. Qin S, Jiang P, Tseng CK. Molecular biotechnology of marine algae in Chaina. Hydrobiologia 2004;512(1-3) 21-26.
  116. 116. Kindle KL, Schnell RA, Fernandez E, Lefebvre PA. Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. Journal of Cell Biology 1989;109(6 Part1) 2589–2601.
  117. 117. Kindle KL. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences of the United States of America 1990;87(3) 1228-1232.
  118. 118. Brown LE, Sprecher SL, Keller LR. Introduction of exogenous DNA into Chlamidomonas reinhardtii by electroporation. Molecular and Cellular Biology 1991;11(4) 2328-2332.
  119. 119. Maruyama M, Horákova I, Honda H, Xing X, Shiragami N, Unno H. Introduction of foreign DNA into Chlorella saccharophila by electroporation. Biotechnology Techniques 1994;8(11) 821-826.
  120. 120. Teng C, Qin S, Liu J, Yu D, Liang C, Tseng C. Transient expression of lacZ in bombarded unicellular green alga Haematococcua pluvialis. Journal of Applied Phycology 2002;14(6) 495-500.
  121. 121. Hallmann A, Rappel A, Sumper M. Gene replacement by homologous recombination in the multicellular green alga Volvox carteri. Proceedings of the National Academy of Sciences of the United States of America 1997;94(14) 7469-7474.
  122. 122. Lerche K, Hallmann A. Stable nuclear transformation of Gonium pectoral. BMC Biotechnol 2009;9 64. (doi:10.1186/1472-6750-9-64) http://www.biomedcentral.com/1472-6750/9/64 (accessed 10 July 2009).
  123. 123. Kathiresan S, Chandrashekar A, Ravishankar GA, Sarada R. Agrobacterium-mediated transformation in the green alga Haematococcus pluvialis (Chlorophyceae, Volvocales). Journal of Phycology 2009;45(3) 642-649.
  124. 124. Huang, X, Weber JC, Hinson TK, Mathieson AC, Minocha SC. Transient expression of the GUS reporter gene in the protoplasts and partially digested cells of Ulva lactuca L (Chlorophyta). Botanica Marina 1996;39(1-6) 467-474.
  125. 125. Kakinuma M, Ikeda M, Coury DA, Tominaga H, Kobayashi I, Amano H. Isolation and characterization of the rbcS genes from a sterile mutant of Ulva pertusa (Ulvales, Chlorophytea) and transient gene expression using the rbcS gene promoter. Fisheries Science 2009;75(4) 1015-1028.
  126. 126. Franklin S, Ngo B, Efuet E, Mayfield SP. Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant Journal 2002;30(6) 733-744.
  127. 127. Ohnuma M, Yokoyama T, Inouye T, Sekine Y, Tanaka K. Polyethylene glycol (PEG)-mediated transient gene expression in a red alga, Cyanidioschyzon merolae 10D. Plant and Cell Physiology 2008;49(1) 117-120.
  128. 128. Zuo Z, Li B, Wang C, Cai J, Chen Y. Increasing transient expression of CAT gene in Porphyra haitanensis by matrix attachment regions and 18S rDNA targeted homologous recombination. Aquaculture Research 2007;38(7) 681-688.
  129. 129. He P, Yao Q, Chen Q , Guo M, Xiong A, Wu W, Ma J. Transferring and expression of glucose oxidase gene in Porphyra yezoensis. Journal of Phycology 2001;37(Supplement) 23.
  130. 130. Mizukami Y, Hado M, Kito H, Kunimoto M, Murase N. Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis. Journal of Applied Phycology 2004;16(1) 23-29.
  131. 131. Wang J, Jiang P, Cui Y, Guan X, Qin S. Gene transfer into conchospores of Porphyra haitanensis (Bangiales, Rhodophyta) by glass bead agitation. Phycologia 2010;49(4) 355-360.

Written By

Koji Mikami

Submitted: March 1st, 2012 Published: July 1st, 2013

© 2013 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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