Abstract
A mutated phytoene desaturase (pds) gene, pds-L504R, conferring resistance to the herbicide norflurazon has been reported as a dominant selectable marker for the genetic engineering of microalgae (Steinbrenner and Sandmann in Appl Environ Microbiol 72:7477–7484, 2006; Prasad et al. in Appl Microbiol Biotechnol 98(20):8629–8639, 2014). However, this mutated genomic clone harbors several introns and the entire expression cassette including its native promoter and terminator has a length > 5.6 kb, making it unsuitable as a standard selection marker. Therefore, we designed a synthetic, short pds gene (syn-pds-int) by removing introns and unwanted internal restriction sites, adding suitable restriction sites for cloning purposes, and introduced the first intron from the Chlamydomonas reinhardtii RbcS2 gene close to the 5′end without changing the amino acid sequence. The syn-pds-int gene (1872 bp) was cloned into pCAMBIA 1380 under the control of a short sequence (615 bp) of the promoter of pds (pCAMBIA 1380-syn-pds-int). This vector and the plasmid pCAMBIA1380-pds-L504R hosting the mutated genomic pds were used for transformation studies. To broaden the existing transformation portfolio, the rhodophyte Porphyridium purpureum was targeted. Agrobacterium-mediated transformation of P. purpureum with both the forms of pds gene, pds-L504R or syn-pds-int, yielded norflurazon-resistant (NR) cells. This is the first report of a successful nuclear transformation of P. purpureum. Transformation efficiency and lethal norflurazon dosage were determined to evaluate the usefulness of syn-pds-int gene and functionality of the short promoter of pds. PCR and Southern blot analysis confirmed transgene integration into the microalga. Both forms of pds gene expressed efficiently as evidenced by the stability, tolerance and the qRT-PCR analysis. The molecular toolkits and transformation method presented here could be used to genetically engineer P. purpureum for fundamental studies as well as for the production of high-value-added compounds.
Similar content being viewed by others
References
Ali S, Xianyin Z, Xue Q, Hassan MJ, Qian H (2007) Investigations for improved genetic transformation mediated by Agrobacterium tumefaciens in two rice cultivars. Biotechnol 6:138–147
Allnutt FCT, Kyle DJ, Grossman AR, Apt KE (2000) Methods and tools for transformation of eukaryotic algae. United States of America, Patent Number, p 6027900
Andersen RA, Berges JA, Harrison PJ, Watanabe MM (2005) Recipes for freshwater and seawater media. In: Andersen RA (ed) Algal culturing techniques. Elsevier, Amsterdam, pp 429–538
Anila N, Chandrashekar A, Ravishankar GA, Sarada R (2011) Establishment of Agrobacterium tumefaciens-mediated genetic transformation in Dunaliella bardawil. Eur J Phycol 46(1):36–44
Bai LL, Yin WB, Chen YH, Niu LL, Sun YR, Zhao SM, Yang FQ, Wang RRC, Wu Q, Zhang XQ, Hu ZM (2013) A new strategy to produce a defensin: stable production of mutated NP-1 in nitrate reductase-deficient Chlorella ellipsoidea. PLoS ONE 8(1):e54966
Barik DP, Mohapatra U, Chand PK (2005) Transgenic grasspea (Lathyrus sativus L.): factors influencing Agrobacterium-mediated transformation and regeneration. Plant Cell Rep 24:523–531
Bhattacharya D, Price DC, Chan CX, Qiu H, Rose N, Ball S, Weber AP, Arias MC, Henrissat B, Coutinho PM, Krishnan A, Zäuner S, Morath S, Hilliou F, Egizi A, Perrineau MM, Yoon HS (2013) Genome of the red alga Porphyridium purpureum. Nat Commun 4:1941
Brodie J, Chan CX, De Clerck O, Cock JM, Coelho SM, Gachon C, Grossman AR, Mock T, Raven JA, Smith AG, Yoon HS (2017) The algal revolution. Trends Plant Sci 22(8):726–738
Bruggeman AJ, Kuehler D, Weeks DP (2014) Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production. Plant Biotechnol J 12:894–902
Cadoret JP, Garnier M, Jean BS (2012) Microalgae, functional genomics and biotechnology. Adv Bot Res 64:285–341
Cha TS, Yee W, Aziz A (2012) Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green algae, Chlorella vulgaris. World J Microbiol Biotechnol 28:1771–1779
Cheng R, Ma R, Li K, Rong H, Lin X, Wang Z, Yang S, Ma Y (2012) Agrobacterium tumefaciens mediated transformation of marine microalgae Schizochytrium. Microbiol Res 167(3):179–186
Chomczynski P, Mackey K (1995) Short technical report. Modification of the TRIZOL reagent procedure for isolation of RNA from Polysaccharide-and proteoglycan-rich sources. Biotechniques 19(6):942–945
Den Dulk-Ras A, Hooykaas PJJ (1995) Electroporation of Agrobacterium tumefaciens. In: Nickoloff JA (ed) Plant cell electroporation and electrofusion protocols (Methods in Molecular Biology). Humana Press, Totowa, pp 63–73
Doron L, Segal N, Shapira M (2016) Transgene expression in microalgae—from tools to applications. Front Plant Sci 7:505
Gangl D, Zedler JAZ, Rajakumar PD, Martinez EMR, Riseley A, Włodarczyk A, Purton S, Sakuragi Y, Howe CJ, Jensen PE, Robinson C (2015) Biotechnological exploitation of microalgae. J Exp Bot 66:6975–6990
García JL, Vicente M, Galan B (2017) Microalgae, old sustainable food and fashion nutraceuticals. Microb Biotechnol 10(5):1017–1024
Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the ‘Gene-Jockeying’ tool. Microbiol Mol Biol Rev 67:16–37
Gong Y, Hu H, Gao Y, Xu X, Gao H (2011) Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects. J Ind Microbiol Biotechnol 38(12):1879–1890
Hlavova M, Turoczy Z, Bisova K (2015) Improving microalgae for biotechnology—from genetics to synthetic biology. Biotechnol Adv 33:1194–1203
Hu Z, Wu Y, Li W, Gao H (2006) Factors affecting Agrobacterium mediated genetic transformation of Lycium barbarum L. Vitro Cell Dev Biol Plant 42:461–466
Jaeger D, Hübner W, Huser T, Mussgnug JH, Kruse O (2017) Nuclear transformation and functional gene expression in the oleaginous microalga Monoraphidium neglectum. J Biotechnol 249:10–15
Jinkerson RE, Jonikas MC (2015) Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome. Plant J 82(3):393–412
Karami O (2008) Factors Affecting Agrobacterium-mediated transformation of plants. Transgenic Plant J 2(2):127–137
Kathiresan S, Chandrashekar A, Ravishankar GA, Sarada R (2009) Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis (Chlorophyceae, Volvocales). J Phycol 45:642–649
Katiyar R, Gurjar BR, Biswas S, Pruthi V, Kumar N, Kumar P (2017) Microalgae: an emerging source of energy based bio-products and a solution for environmental issues. Renew Sust Energ Rev 72:1083–1093
Kavitha MD, Kathiresan S, Bhattacharya S, Sarada R (2016) Culture media optimization of Porphyridium purpureum: production potential of biomass, total lipids, arachidonic and eicosapentaenoic acid. J Food Sci Technol 53(5):2270–2278
Kovar JL, Zhang J, Funke RP, Weeks DP (2002) Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation. Plant J 29:109–117
Kumar SV, Misquitta RW, Reddy VS, Rao BJ, Rajam MV (2004) Genetic transformation of the green alga-Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Sci 166:731–738
Kumar S, Raja SK, Sharmab AK, Varmac HN (2012) Genetic transformation and development of Cucumber mosaic virus resistant transgenic plants of Chrysanthemum morifolium cv. Kundan. Sci Hortic 134:40–45
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
León R, Fernandez E (2007) Nuclear transformation of eukaryotic microalgae: historical overview, achievements and problems. Adv Exp Med Biol 616:1–11
Liu J, Zhong Y, Sun Z, Huang J, Sandmann G, Chen F (2010) One amino acid substitution in phytoene desaturase makes Chlorella zofingiensis resistant to norflurazon and enhances the biosynthesis of astaxanthin. Planta 232(1):61–67
Liu J, Gerken H, Huang J, Chen F (2013) Engineering of an endogenous phytoene desaturase gene as a dominant selectable marker for Chlamydomonas reinhardtii transformation and enhanced biosynthesis of carotenoids. Proc Biochem 48:788–795
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408
Lumbreras V, Stevens DR, Purton S (1998) Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J 14:441–447
Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S, Lee YC (2005) Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem 339(1):69–72
Mussgnug JH (2015) Genetic tools and techniques for Chlamydomonas reinhardtii. Appl Microbiol Biotechnol 99(13):5407–5418
Muto M, Fukuda Y, Nemoto M, Yoshino T, Matsunaga T, Tanaka T (2013) Establishment of a genetic transformation system for the marine pennate diatom Fistulifera sp. strain JPCC DA0580—a high triglyceride producer. Mar Biotechnol 15:48–55
Naing AH, Ai TN, Jeon SM, Lim SH, Kim CK (2016) An efficient protocol for Agrobacterium-mediated genetic transformation of recalcitrant chrysanthemum cultivar Shinma. Acta Physiol Plant 38(2):38
Ng I-S, Tan S-I, Kao P-H, Chang Y-K, Chang J-S (2017) Recent developments on genetic engineering of microalgae for biofuels and bio-based chemicals. Biotechnol J. https://doi.org/10.1002/biot.201600644
Noda J, Mühlroth A, Bučinská L, Dean J, Bones AM, Sobotka R (2017) Tools for biotechnological studies of the freshwater alga Nannochloropsis limnetica: antibiotic resistance and protoplast production. J Appl Phycol 29(2):853–863
Oudot-Le Secq M-P, Grimwood J, Shapiro H, Armbrust EV, Bowler C, Green BV (2007) Chloroplast genomes of the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana: comparison with other plastid genomes of the red lineage. Mol Genet Genomics 277(4):427–439
Prasad B, Nithya V, Jeong HJ, General T, Cho M-Gi, Lein W (2014) Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species). Appl Microbiol Biotechnol 98(20):8629–8639
Prasad B, Lein W, Lindenberger CP, Buchholz R, Vadakedath N (2018) An optimized method and a dominant selectable marker for genetic engineering of an industrially promising microalga—Pavlova lutheri. J Appl Phycol. https://doi.org/10.1007/s10811-018-1617-9
Pratheesh PT, Vineetha M, Kurup GM (2014) An efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii. Mol Biotechnol 56(6):507–515
Purton S, Szaub JB, Wannathong T, Young R, Economou CK (2013) Genetic engineering of algal chloroplasts: progress and prospects. Rus J Plant Physiol 60(4):521–528
Rathod JP, Prakash G, Pandit R, Lali AM (2013) Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri. Photosynth Res 118(1):141–146
Reddy PH, Johnson AMA, Kumar JK, Naveen T, Devi MC (2017) Heterologous expression of Infectious bursal disease virus VP2 gene in Chlorella pyrenoidosa as a model system for molecular farming. Plant Cell Tissue Organ Cult 131(1):119–126
Sato N, Moriyama T, Mori N, Toyoshima M (2017) Lipid metabolism and potentials of biofuel and high added-value oil production in red algae. World J Microbiol Biotechnol 33(4):74
Sharon-Gojman R, Maimon E, Leu S, Zarka A, Boussiba S (2015) Advanced methods for genetic engineering of Haematococcus pluvialis (Chlorophyceae, Volvocales). Algal Res 10:8–15
Simon DP, Anila N, Gayathri K, Sarada R (2016) Heterologous expression of β-carotene hydroxylase in Dunaliella salina by Agrobacterium-mediated genetic transformation. Algal Res 18:257–265
Srinivasan R, Gothandam KM (2016) Synergistic action of D-glucose and acetosyringone on Agrobacterium strains for efficient Dunaliella transformation. PLoS ONE 11(6):e0158322
Steinbrenner J, Sandmann G (2006) Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis. Appl Environ Microbiol 72:7477–7484
Úbeda-Mínguez P, Chileh T, Dautor Y, García-Maroto F, Alonso DL (2015) Tools for microalgal biotechnology: development of an optimized transformation method for an industrially promising microalga-Tetraselmis chuii. J Appl Phycol 27(1):223–232
Velea S, Ilie L, Filipescu L (2011) Optimization of Porphyridium purpureum culture growth using two variables experimental design: light and sodium bicarbonate. UPB Sci Bull Series B 73(4):81–94
Wang C, Wang Y, Su Q, Gao X (2007) Transient expression of the GUS gene in a unicellular marine green alga, Chlorella sp. MACC/C95, via electroporation. Biotechnol Bioprocess Eng 12:180–183
Wei X, Chen C, Yu Q, Gady A, Yu Y, Liang G, Gmitter FG Jr (2014) Comparison of carotenoid accumulation and biosynthetic gene expression between Valencia and Rohde Red Valencia sweet oranges. Plant Sci 227:28–36
Acknowledgements
The authors are thankful to Dr. J. Steinbrenner (Universität Konstanz, Germany) and Prof. Choi PS (Nambu University, South Korea) for kindly providing the plasmid pPLAT-pds-L504R and Agrobacterium tumefaciens strain LBA4404, respectively. The authors also appreciate the funding bodies Korea Institute of Advanced Technology, Korea, and the Federal Ministry of Education and Research, Germany for supporting the work. BP also acknowledges Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-University of Erlangen Nuremberg, Germany for the research support.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
All authors declare that they have no conflict of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Prasad, B., Lein, W., Thiyam, G. et al. Stable nuclear transformation of rhodophyte species Porphyridium purpureum: advanced molecular tools and an optimized method. Photosynth Res 140, 173–188 (2019). https://doi.org/10.1007/s11120-018-0587-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11120-018-0587-8