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Construction of Plastid Expression Vector and Development of Genetic Transformation System for the Seaweed Pyropia yezoensis

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Abstract

Pyropia yezoensis, belonging to the Rhodophyta, is an economically important seaweed. In this study, we developed a high-efficiency plastid transformation platform for P. yezoensis. In the plastid transformation vector, psbA UTR of P. yezoensis, including the promoter and 3′ UTR, was used to express foreign genes. The integration site was a transcriptionally active intergenic region between the rrsB and trnI genes, located in the inverted repeat regions of the plastid genome. The CAT and eGFP genes were integrated into the plastid genome at this site. The expression of CAT in the transformants confers resistance to chloramphenicol through the action of chloramphenicol acetyltransferase, which inactivates the drug, thereby allowing the plant to grow well under selective pressure. The eGFP fluorescence signal was also observed in transformed cells and the transformants. The average survival rate of treated cells was estimated to be approximately 4.2‰ (4 transplastomic colonies per 1000 gametophyte cells). Multiple-PCR analyses confirmed that the CAT and eGFP genes were successfully integrated in the site between rrsB and trnI. Western blot also showed eGFP expression in the cells of transformants. Thus, this study presents the first convenient plastid gene expression system for P. yezoensis and provides an important platform for studying gene function in P. yezoensis.

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References

  • Bertalan I, Munder MC, Weiss C, Kopf J, Fischer D, Johanningmeier U (2015) A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii. J Biotechnol 195:60–66

    Article  CAS  PubMed  Google Scholar 

  • Blouin NA, Brodie JA, Grossman AC, Xu P, Brawley SH (2010) Porphyra: a marine crop shaped by stress. Trends Plant Sci 16:29–37

    Article  PubMed  Google Scholar 

  • Bock R (2015) Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology. Annu Rev Plant Biol 66:211–241

    Article  CAS  PubMed  Google Scholar 

  • Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein TM, Shark KB et al (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240:1534–1538

    Article  CAS  PubMed  Google Scholar 

  • Chaudhuri S, Carrer H, Maliga P (1995) Site-specific factor involved in the editing of the psbL mRNA in tobacco plastids. EMBO J 14:2951–2957

    CAS  PubMed  PubMed Central  Google Scholar 

  • Daniell H, Khan MS, Allison L (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci 7:84–91

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Daniell H, Kumar S, Dufourmantel N (2005) Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends Biotechnol 23:238–245

  • Dauvillee D, Hilbig L, Preiss S, Johanningmeier U (2004) Minimal extent of sequence homology required for homologous recombination at the psbA locus in Chlamydomonas reinhardtii chloroplasts using PCR-generated DNA fragments. Photosynth Res 79:219–224

    Article  CAS  PubMed  Google Scholar 

  • Davison IR, Pearson GA (1996) Stress tolerance in intertidal seaweeds. J Phycol 32:197–211

    Article  Google Scholar 

  • Day A, Goldschmidt-Clermont M (2011) The chloroplast transformation toolbox: selectable markers and marker removal. Plant Biotechnol J 9:540–553

    Article  CAS  PubMed  Google Scholar 

  • De Cosa B, Moar W, Lee SB, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19:71–74

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Doetsch NA, Favreau MR, Kuscuoglu N, Thompson MD, Hallick RB (2001) Chloroplast transformation in Euglena gracilis: splicing of a group III twintron transcribed from a transgenic psbK operon. Curr Genet 39:49–60

    Article  CAS  PubMed  Google Scholar 

  • Dufourmantel N, Pelissier B, Garcon F, Peltier G, Ferullo JM, Tissot G (2004) Generation of fertile transplastomic soybean. Plant Mol Biol 55:479–489

    Article  CAS  PubMed  Google Scholar 

  • Fischer N, Stampacchia O, Redding K, Rochaix JD (1996) Selectable marker recycling in the chloroplast. Mol Gen Genet 251:373–380

    Article  CAS  PubMed  Google Scholar 

  • Hirata R, Takahashi M, Saga N, Mikami K (2011) Transient gene expression system established in Porphyra yezoensis is widely applicable in Bangiophycean algae. Mar Biotechnol (NY) 13:1038–1047

    Article  CAS  Google Scholar 

  • Iamtham S, Day A (2000) Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat Biotechnol 18:1172–1176

    Article  CAS  PubMed  Google Scholar 

  • Kanamoto H, Yamashita A, Asao H, Okumura S, Takase H, Hattori M, Yokota A, Tomizawa K (2006) Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res 15:205–217

    Article  CAS  PubMed  Google Scholar 

  • Katsuhara M, Koshio K, Shibasaka M, Hayashi Y, Hayakawa T, Kasamo K (2003) Over-expression of a barley aquaporin increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants. Plant Cell Physiol 44:1378–1383

    Article  CAS  PubMed  Google Scholar 

  • Kittiwongwattana C, Lutz K, Clark M, Maliga P (2007) Plastid marker gene excision by the phiC31 phage site-specific recombinase. Plant Mol Biol 64:137–143

  • Lapidot M, Raveh D, Sivan A, Arad SM, Shapira M (2002) Stable chloroplast transformation of the unicellular red alga Porphyridium species. Plant Physiol 129:7–12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li W, Ruf S, Bock R (2010) Chloramphenicol acetyltransferase as selectable marker for plastid transformation. Plant Mol Biol 76:443–451

  • Lilly JW, Havey MJ, Jackson SA, Jiang J (2001) Cytogenomic analyses reveal the structural plasticity of the chloroplast genome in higher plants. Plant Cell 13:245–254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu CW, Lin CC, Chen JJ, Tseng MJ (2007) Stable chloroplast transformation in cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment. Plant Cell Rep 26:1733–1744

    Article  CAS  PubMed  Google Scholar 

  • Lutz KA, Bosacchi MH, Maliga P (2006) Plastid marker-gene excision by transiently expressed CRE recombinase. Plant J 45:447–456

    Article  CAS  PubMed  Google Scholar 

  • Meyers B, Zaltsman A, Lacroix B, Kozlovsky SV, Krichevsky A (2010) Nuclear and plastid genetic engineering of plants: comparison of opportunities and challenges. Biotechnol Adv 28:747–756

    Article  CAS  PubMed  Google Scholar 

  • Millán AFS, Mingo-Castel A, Miller M,Daniell H (2003) A chloroplast transgenic approach to hyper-express and purify HumanSerum Albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnol J 1:71–79

  • Muhlbauer SK, Koop HU (2005) External control of transgene expression in tobacco plastids using the bacterial lac repressor. Plant J 43:941–946

    Article  PubMed  Google Scholar 

  • Muto M, Henry RE, Mayfield SP (2009) Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast. BMC Biotechnol 9:26

    Article  PubMed  PubMed Central  Google Scholar 

  • Nugent GD, Ten Have M, Van Der Gulik A, Dix PJ, Uijtewaal BA, Mordhorst AP (2005) Plastid transformants of tomato selected using mutations affecting ribosome structure. Plant Cell Rep 24:341–349

    Article  CAS  PubMed  Google Scholar 

  • Oey M, Lohse M, Kreikemeyer B, Bock R (2009) Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J 57:436–445

    Article  CAS  PubMed  Google Scholar 

  • Rasala BA, Mayfield SP (2015) Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses. Photosynth Res 123:227–239

    Article  CAS  PubMed  Google Scholar 

  • Sahoo D, Tang X, Yarish C (2002) Porphyra—the economic seaweed as a new experimental system. Curr Sci 83:1313–1316

    Google Scholar 

  • Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PT, Staub JM, Nehra NS (1999) Technical Advance: Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J 19:209–216

    Article  CAS  PubMed  Google Scholar 

  • Sugiura C, Sugita M (2004) Plastid transformation reveals that moss tRNA(Arg)-CCG is not essential for plastid function. Plant J 40:314–321

    Article  CAS  PubMed  Google Scholar 

  • Takahashi M, Koji M, Hiroyuki M, Naotsune S (2011) Identification and efficient utilization of antibiotics for the development of a stable transformation system in Porphyra yezoensis (Bangiales, Rhodophyta). J Aquac Res Dev S3. doi:10.4172/2155-9546.S3-002

  • Uji T, Hirata R, Fukuda S, Mizuta H, Saga N (2014) A codon-optimized bacterial antibiotic gene used as selection marker for stable nuclear transformation in the marine red alga Pyropia yezoensis. Mar Biotechnol (NY) 16:251–255

    Article  CAS  Google Scholar 

  • Verma D, Daniell H (2007) Chloroplast vector systems for biotechnology applications. Plant Physiol 145:1129–1143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang HH, Yin WB, Hu ZM (2009) Advances in chloroplast engineering. J Genet Genomics 36:387–398

    Article  CAS  PubMed  Google Scholar 

  • Wang L, Mao Y, Kong F, Li G, Ma F, Zhang B, Sun P, Bi G, Zhang F, Xue H, Cao M (2013) Complete sequence and analysis of plastid genomes of two economically important red algae: Pyropia haitanensis and Pyropia yezoensis. PLoS One 8:e65902

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Watson J, Koya V, Leppla SH, Daniell H (2004) Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop. Vaccine 22:4374–4384

  • Xie WH, Zhu CC, Zhang NS, Li DW, Yang WD, Liu JS, Sathishkumar R, Li HY (2014) Construction of novel chloroplast expression vector and development of an efficient transformation system for the diatom Phaeodactylum tricornutum. Mar Biotechnol (NY) 16:538–546

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zoubenko OV, Allison LA, Svab Z, Maliga P (1994) Efficient targeting of foreign genes into the tobacco plastid genome. Nucleic Acids Res 22:3819–3824

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Acknowledgements

This work was supported by Natural Science Foundation of China (grant no. 31672641) and National High Technology Research and Development Program of China (grant no. 2012AA10A401). We appreciate that Dr. John van der Meer assisted with manuscript editing.

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Authors and Affiliations

  1. College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China

    Fanna Kong, Hailong Zhao, Weixun Liu, Na Li & Yunxiang Mao

  2. Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao, 266003, China

    Fanna Kong, Hailong Zhao, Weixun Liu, Na Li & Yunxiang Mao

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  1. Fanna Kong
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  2. Hailong Zhao
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  3. Weixun Liu
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  4. Na Li
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Correspondence to Fanna Kong or Yunxiang Mao.

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Kong, F., Zhao, H., Liu, W. et al. Construction of Plastid Expression Vector and Development of Genetic Transformation System for the Seaweed Pyropia yezoensis . Mar Biotechnol 19, 147–156 (2017). https://doi.org/10.1007/s10126-017-9736-x

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