Transient Transformation of Red Algal Cells: Breakthrough Toward Genetic Transformation of Marine Crop Porphyra Species

Mikami, Koji; Hirata, Ryo; Takahashi, Megumu; Uji, Toshiki; Saga, Naotsune

doi:10.5772/20982

Cited by 17 publications

(31 citation statements)

References 68 publications

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“…However, we have found that the TATA box is not usually found in the core promoters of P. yezoensis genes (unpublished observation), and we thus proposed that there were differences in the promoter structure and transcriptional regulation of protein-coding genes between red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][38][39][40][41].…”

Section: Recent Improvement Of the Transient Transformation System Incontrasting

confidence: 59%

“…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].…”

Section: Optimization Of Codon Usage In the Reporter Genementioning

confidence: 99%

“…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 An Integrated View of the Molecular Recognition and Toxinology -From Analytical Procedures to Biomedical Applications [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.…”

Section: Introductionmentioning

confidence: 99%

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Current Advances in Seaweed Transformation

Mikami¹

2013

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical Applications

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Section: Recent Improvement Of the Transient Transformation System Incontrasting

confidence: 59%

Section: Optimization Of Codon Usage In the Reporter Genementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Current Advances in Seaweed Transformation

Mikami¹

2013

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical Applications

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“…To date, several studies have described the establishment of a transient gene expression system in P. yezoensis [9]; for example, we have succeeded in eliciting the efficient expression of the codon-modified β-glucuronidase (PyGUS) and humanized cyan and green fluorescent protein (AmCFP and ZsGFP) genes by transient transformation of P. yezoensis gametophytes using particle bombardment [10][11][12][13], which was then adapted to other Bangiophycean algae [14,15]. Despite the development of transient gene expression systems in P. yezoensis, a stable transformation system, which is needed for the fine analysis of gene function, has not been established, because of a lack of techniques to select and isolate stably transformed cells from gametophytes.…”

Section: Introductionmentioning

confidence: 99%

Identification and Efficient Utilization of Antibiotics for the Development of a Stable Transformation System in Porphyra yezoensis (Bangiales,Rhodophyta)

Mikami¹

2014

J Aquac Res Development

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IntroductionMicroalgae and macroalgae are of great value both as organisms for basic biological research and as resources for bio-industry. That is why algae are now considered to be very promising organisms for economical and industrial applications and are the target of genetic transformation [1,2]. Recently, such transformation has been performed successfully in microalgae, and stably transformed microalgae have been employed to produce useful materials and to analyze the gene functions required for engineering these materials [3][4][5]. However, it is still hard to establish transgenic macroalgae, which is hindering our understanding of gene functions in various physiological regulations and also slowing our utilization of macroalgae in biotechnological applications.The red macroalga Porphyra yezoensis is the most popular sea crop in Asia [6] and has recently received a great deal of attention as a model macrophyte for physiological and molecular biological studies in marine red algae [7,8]. To date, several studies have described the establishment of a transient gene expression system in P. yezoensis [9]; for example, we have succeeded in eliciting the efficient expression of the codon-modified β-glucuronidase (PyGUS) and humanized cyan and green fluorescent protein (AmCFP and ZsGFP) genes by transient transformation of P. yezoensis gametophytes using particle bombardment [10][11][12][13], which was then adapted to other Bangiophycean algae [14,15]. Despite the development of transient gene expression systems in P. yezoensis, a stable transformation system, which is needed for the fine analysis of gene function, has not been established, because of a lack of techniques to select and isolate stably transformed cells from gametophytes.The availability of a useful selection protocol, which depends on the choice of a selection agent such as antibiotics and herbicides, is generally a prerequisite for the development of a stable transformation system [16]. Thus, the identification of selection agents such as antibiotics and herbicides was carried out in P. yezoensis [17], and it resulted in the indication of the non-sensitivity of P. yezoensis gametophytes to many kinds of antibiotics at concentrations up to 5 mg ml-1 in culture medium; however, the reproducibility of the test results was not examined. Therefore, we here identified a selection agent for a stable transformation system in P. yezoensis gametophytes by reexamining the sensitivity to 4 antibiotics used in the previous study, ampicillin, kanamycin, hygromycin B and geneticin [17], as well as that of chloramphenicol and paromomycin. In addition, since the high salt conditions often reduced the action of antibiotics in many marine algae [18] and high pH levels of the medium affected the toxicity of the antibiotic trimethoprim (TMP) for the willow tree Salix viminalis [19], we also investigated using an artificial synthetic medium to determine whether modification of the medium conditions in terms of pH value and salt concentration would affect the antibi...

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“…To date, there is no way to analyze the function of PyKPA1 using P. yezoensis, because of the lack of genetic manipulation systems in any seaweed for the inactivation or knockdown of genes Mikami et al 2011). Therefore, heterologous expression is only means of functional analysis of PyKPA1.…”

mentioning

confidence: 99%

Functional expression of an animal type-Na+-ATPase gene from a marine red seaweed Porphyra yezoensis increases salinity tolerance in rice plants

Kishimoto

Shimajiri

Oshima

et al. 2013

Plant Biotechnology

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Despite lacking Na + -ATPase as a sodium pump in vascular plants, a gene encoding KPA (K + P-type ATPase), a putative animal type-Na + /K + -ATPase, has been isolated from the marine red alga Porphyra yezoensis and designated PyKPA1. To characterize the properties of PyKPA1 and also to confirm its ability to confer salinity tolerance in land plants, transgenic rice plants were produced that expressed the full-length PyKPA1 cDNA under the control of cauliflower mosaic virus 35S RNA promoter. We observed transcriptional activation of the transgene, plasma membrane-localization of the gene product fused with green fluorescent protein in onion epidermal cells, and Na + -ATPase activity in the plasma membrane fraction from transgenic rice plants, indicating that PyKPA1 was functionally expressed in rice plants. Transgenic lines were examined in terms of growth in salinity stress conditions, resulting in protection from a decrease in biomass, although growth of control rice plants was repressed. These results demonstrate the utility of a red algal animal type-sodium pump for conferring salinity tolerance to land plants.

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Transient Transformation of Red Algal Cells: Breakthrough Toward Genetic Transformation of Marine Crop Porphyra Species

Cited by 17 publications

References 68 publications

Current Advances in Seaweed Transformation

Current Advances in Seaweed Transformation

Identification and Efficient Utilization of Antibiotics for the Development of a Stable Transformation System in Porphyra yezoensis (Bangiales,Rhodophyta)

Functional expression of an animal type-Na+-ATPase gene from a marine red seaweed Porphyra yezoensis increases salinity tolerance in rice plants

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