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The pollen- and embryo-specific Arabidopsis DLL promoter bears good potential for application in marker-free Cre/loxP self-excision strategy

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Abstract

Key message

Marker-free transgenic plants can be generated with high efficiency by using the Cre/ lox P self-excision system controlled by the pollen- and embryo-specific Arabidopsis DLL promoter.

Abstract

In this work, we aimed to study the feasibility of using the pollen- and embryo-specific DLL promoter of the At4g16160 gene from Arabidopsis thaliana in a Cre/loxP self-excision strategy. A Cre/loxP self-excision cassette controlled by the DLL promoter was introduced into the tobacco genome via Agrobacterium-mediated transformation. No evidence for premature activation of the Cre/loxP system was observed in primary transformants. The efficiency of nptII removal during pollen and embryo development was investigated in transgenic T1 progenies derived from eight self- and four cross-pollinated T0 lines, respectively. Segregation and rooting assays were performed to select recombined T1 plants. Molecular analyses of these plants confirmed the excision event in all analysed T0 lines and marker-free transgenic T1 plants were obtained with efficiency of up to 96.2 %. The Arabidopsis DLL promoter appears to be a strong candidate to drive Cre-mediated recombination not only in tobacco as a model plant, but also in other plant species.

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References

  • Bai X, Wang Q, Chu C (2008) Excision of a selective marker in transgenic rice using a novel Cre/loxP system controlled by a floral specific promoter. Transgenic Res 17:1035–1043

    Article  CAS  PubMed  Google Scholar 

  • Boszorádová E, Libantová J, Matušíková I, Moravčíková J (2014) Application of Arabidopsis tissue-specific CRUC promoter in the Cre/loxP self-excision strategy for generation of marker-free oilseed rape: potential advantages and drawbacks. Acta Physiol Plant 36:1399–1409

    Article  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chakraborti D, Sarkar A, Mondal HA, Schuermann D, Hohn B, Sarmah BK, Das S (2008) Cre/lox system to develop selectable marker-free transgenic tobacco plants conferring resistance against sap sucking homopteran insect. Plant Cell Rep 27:1623–1633

    Article  CAS  PubMed  Google Scholar 

  • Chen J, Greenblatt IM, Dellaporta SL (1992) Molecular analysis of Ac transposition and DNA-replication. Genetics 130:665–676

    PubMed Central  CAS  PubMed  Google Scholar 

  • Chong-Perez B, Reyes M, Rojas L, Ocana B, Ramos A, Kosky RG, Angenon G (2013) Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter. Plant Mol Biol 83:143–152

    Article  CAS  PubMed  Google Scholar 

  • Coppoolse ER, de Vroomen MJ, Roelofs D, Smit J, van Gennip F, Hersmus BJM, Nijkamp HJJ, van Haaren MJJ (2003) Cre recombinase expression can result in phenotypic aberrations in plants. Plant Mol Biol 51:263–279

    Article  CAS  PubMed  Google Scholar 

  • Costa-Font M, Gil JM, Traill WB (2008) Consumer acceptance, valuation of and attitudes towards genetically modified food: review and implications for food policy. Food Policy 33:99–111

    Article  Google Scholar 

  • Cuellar W, Gaudin A, Solorzano D, Casas A, Nopo L, Chudalayandi P, Medrano G, Kreuze J, Ghislain M (2006) Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre–loxP system from transgenic potato. Plant Mol Biol 62:71–82

    Article  CAS  PubMed  Google Scholar 

  • Dale EC, Ow DW (1991) Gene-transfer with subsequent removal of the selection gene from the host genome. P Natl Acad Sci USA 88:10558–10562

    Article  CAS  Google Scholar 

  • Drea SC, Lao NT, Wolfe KH, Kavanagh TA (2006) Gene duplication, exon gain and neofunctionalization of OEP16-related genes in land plants. Plant J 46:723–735

    Article  CAS  PubMed  Google Scholar 

  • García-Almodóvar R, Petri C, Padilla I, Burgos L (2014) Combination of site-specific recombination and a conditional selective marker gene allows for the production of marker-free tobacco plants. Plant Cell Tiss Organ Cult 116:205–215

    Article  Google Scholar 

  • Gidoni D, Srivastava V, Carmi N (2008) Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants. In Vitro Cell Dev Biol Plant 44:457–467

    Article  CAS  Google Scholar 

  • Gilbertson L (2003) Cre–lox recombination: cre-active tools for plant biotechnology. Trends Biotechnol 21:550–555

    Article  CAS  PubMed  Google Scholar 

  • Hoa TTC, Bong BB, Huq E, Hodges TK (2002) Cre/lox site-specific recombination controls the excision of a transgene from the rice genome. Theor Appl Genet 104:518–525

    Article  CAS  PubMed  Google Scholar 

  • Honys D, Twell D (2004) Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol 5:R85

    Article  PubMed Central  PubMed  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) Gus fusions—beta-glucuronidase as a sensitive and versatile gene fusion marker in higher-plants. EMBO J 6:3901–3907

    PubMed Central  CAS  PubMed  Google Scholar 

  • Jopcik M, Bauer M, Moravcikova J, Boszoradova E, Matusikova I, Libantova J (2013) Plant tissue-specific promoters can drive gene expression in Escherichia coli. Plant Cell Tiss Organ Cult 113:387–396

    Article  CAS  Google Scholar 

  • Jopcik M, Moravcikova J, Matusikova I, Libantova J (2014) Spacer length-dependent protection of specific activity of pollen and/or embryo promoters from influence of CaMV 35S promoter/enhancer in transgenic plants. Plant Cell Tiss Organ Cult 118:507–518

  • Kopertekh L, Schiemann J (2005) Agroinfiltration as a tool for transient expression of cre recombinase in vivo. Transgenic Res 14:793–798

    Article  CAS  PubMed  Google Scholar 

  • Kopertekh L, Broer I, Schiemann J (2009) Developmentally regulated site-specific marker gene excision in transgenic B. napus plants. Plant Cell Rep 28:1075–1083

    Article  CAS  PubMed  Google Scholar 

  • Kopertekh L, Saint Paul V, Krebs E, Schiemann J (2012) Utilization of PVX-Cre expression vector in potato. Transgenic Res 21:645–654

    Article  CAS  PubMed  Google Scholar 

  • Li Z, Xing A, Moon BP, Burgoyne SA, Guida AD, Liang H, Lee C, Caster CS, Barton JE, Klein TM, Falco SC (2007) A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants. Plant Mol Biol 65:329–341

    Article  CAS  PubMed  Google Scholar 

  • Liu HK, Yang C, Wei ZM (2005) Heat shock-regulated site-specific excision of extraneous DNA in transgenic plants. Plant Sci 168:997–1003

    Article  CAS  Google Scholar 

  • Luo K, Duan H, Zhao D, Zheng X, Deng W, Chen Y, Stewart CN Jr, McAvoy R, Jiang X, Wu Y, He A, Pei Y, Li Y (2007) GM-gene-deletor: fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotech J 5:263–274

    Article  CAS  Google Scholar 

  • Ma BG, Duan XY, Ma CX, Niu JX, Zhang HP, Pan LZ (2008) Salicylic-acid-induced self-excision of the marker gene nptII from transgenic tomato using the Cre–loxP system. Plant Mol Biol Rep 26:199–212

    Article  CAS  Google Scholar 

  • Ma BG, Duan XY, Niu JX, Ma C, Hao QN, Zhang LX, Zhang HP (2009) Expression of stilbene synthase gene in transgenic tomato using salicylic acid-inducible Cre/loxP recombination system with self-excision of selectable marker. Biotechnol Lett 31:163–169

    Article  CAS  PubMed  Google Scholar 

  • Marjanac G, De Paepe A, Peck I, Jacobs A, De Buck S, Depicker A (2008) Evaluation of CRE-mediated excision approaches in Arabidopsis thaliana. Transgenic Res 17:239–250

    Article  CAS  PubMed  Google Scholar 

  • Matzke AJM, Matzke MA (1998) Position effects and epigenetic silencing of plant transgenes. Curr Opin Plant Biol 1:142–148

    Article  CAS  PubMed  Google Scholar 

  • Mlynarova L, Nap JP (2003) A self-excising Cre recombinase allows efficient recombination of multiple ectopic heterospecific lox sites in transgenic tobacco. Transgenic Res 12:45–57

    Article  CAS  PubMed  Google Scholar 

  • Mlynarova L, Loonen A, Heldens J, Jansen RC, Keizer P, Stiekema WJ, Nap JP (1994) Reduced position effect in mature transgenic plants conferred by the chicken lysozyme matrix-associated region. Plant Cell 6:417–426

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Mlynarova L, Conner AJ, Nap JP (2006) Directed microspore-specific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes. Plant Biotech J 4:445–452

    Article  CAS  Google Scholar 

  • Moravcikova J, Vaculkova E, Bauer M, Libantova J (2008) Feasibility of the seed specific cruciferin C promoter in the self excision Cre/loxP strategy focused on generation of marker-free transgenic plants. Theor Appl Genet 117:1325–1334

    Article  CAS  PubMed  Google Scholar 

  • Murashige I, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 15:473–497

    Article  CAS  Google Scholar 

  • Nicolia A, Manzo A, Veronesi F, Rosellini D (2014) An overview of the last 10 years of genetically engineered crop safety research. Crit Rev in Biotechnol 34:77–88

    Article  CAS  Google Scholar 

  • Odell J, Caimi P, Sauer B, Russell S (1990) Site-directed recombination in the genome of transgenic tobacco. Mol Gen Genet 223:369–378

    Article  CAS  PubMed  Google Scholar 

  • Odell JT, Hoopes JL, Vermerris W (1994) Seed-specific gene activation mediated by the Cre/lox site-specific recombination system. Plant Physiol 106:447–458

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ow DW (2007) GM maize from site-specific recombination technology, what next? Curr Opin in Biotech 18:115–120

    Article  CAS  Google Scholar 

  • Petri C, Lopez-Noguera S, Wang H, Garcia-Almodovar C, Alburquerque N, Burgos L (2012) A chemical-inducible Cre–LoxP system allows for elimination of selection marker genes in transgenic apricot. Plant Cell Tissue Organ Cult 110:337–346

    Article  CAS  Google Scholar 

  • Polóniová Z, Jopčík M, Matušíková I, Libantová J, Moravčíková J (2012) Preparation of plant transformation vector containing “self-excision” Cre/loxP system. J Microbiol Biotech Food Sci 1:563–572

    Google Scholar 

  • Roy SD, Saxena M, Bhomkar PS, Pooggin M, Hohn T, Bhalla-Sarin N (2008) Generation of marker-free salt tolerant transgenic plants of Arabidopsis thaliana using the gly I gene and cre gene under inducible promoters. Plant Cell Tiss Org 95:1–11

    Article  CAS  Google Scholar 

  • Russell SH, Hoopes JL, Odell JT (1992) Directed excision of a transgene from the plant genome. Mol Gen Genet 234:49–59

    CAS  PubMed  Google Scholar 

  • Scutt CP, Zubko E, Meyer P (2002) Techniques for the removal of marker genes from transgenic plants. Biochimie 84:1119–1126

    Article  CAS  PubMed  Google Scholar 

  • Sreekala C, Wu L, Gu K, Wang D, Tian D, Yin Z (2005) Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system. Plant Cell Rep 24:86–94

    Article  CAS  PubMed  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL-W—improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Tuteja N, Verma S, Sahoo RK, Raveendar S, Reddy INBL (2012) Recent advances in development of marker-free transgenic plants: regulation and biosafety concern. Journal Biosci 37:167–197

    Article  CAS  Google Scholar 

  • Vaculkova E, Moravcikova J, Matusikova I, Bauer M, Libantova J (2007) A modified low copy number binary vector pUN for Agrobacterium-mediated plant transformation. Biol Plantarum 51:538–540

    Article  CAS  Google Scholar 

  • Verweire D, Verleyen K, De Buck S, Claeys M, Angenon G (2007) Marker-free Transgenic plants through genetically programmed auto-excision. Plant Physiol 145:1220–1231

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wang Y, Chen BJ, Hu YL, Li JF, Lin ZP (2005) Inducible excision of selectable marker gene from transgenic plants by the Cre/lox site-specific recombination system. Transgen Res 14:605–614

    Article  CAS  Google Scholar 

  • Will E, Klump H, Heffner N, Schwieger M, Schiedlmeier B, Ostertag W, Baum C, Stocking C (2002) Unmodified Cre recombinase crosses the membrane. Nucl Acid Res 30(12):e59

    Article  Google Scholar 

  • Woo H-J, Suh S-C, Cho Y-G (2011) Strategies for developing marker-free transgenic plants. Biotechnol Bioprocess Eng 16:1053–1064

    Article  CAS  Google Scholar 

  • Zhang W, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V, Gilbertson L (2003) Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor Appl Genet 107:1157–1168

    Article  CAS  PubMed  Google Scholar 

  • Zhang YY, Li HX, Bo OY, Lu YG, Ye ZB (2006) Chemical-induced autoexcision of selectable markers in elite tomato plants transformed with a gene conferring resistance to lepidopteran insects. Biotechnol Lett 28:1247–1253

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Liu H, Li B, Zhang JT, Li YZ, Zhang HX (2009) Generation of selectable marker-free transgenic tomato resistant to drought, cold and oxidative stress using the Cre/loxP DNA excision system. Transgen Res 18:607–619

    Article  CAS  Google Scholar 

  • Zuo JR, Niu QW, Moller SG, Chua NH (2001) Chemical-regulated, site-specific DNA excision in transgenic plants. Nat Biotechnol 19:157–161

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

Authors thank Anna Fábelová for in vitro plant care. Funding was supported by Scientific Grant Agency of the Ministry of Education of Slovak Republic and Slovak Academy of Sciences VEGA 2-0090-14.

Conflict of interest

The authors declare they have no conflict of interest.

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Authors and Affiliations

  1. Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, P.O. Box 39A, 95 007, Nitra, Slovak Republic

    Zuzana Polóniová, Martin Jopčík, Ildikó Matušíková, Jana Libantová & Jana Moravčíková

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  1. Zuzana Polóniová
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  2. Martin Jopčík
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  3. Ildikó Matušíková
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  4. Jana Libantová
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  5. Jana Moravčíková
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Corresponding author

Correspondence to Jana Moravčíková.

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Communicated by Zeng-Yu Wang.

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Polóniová, Z., Jopčík, M., Matušíková, I. et al. The pollen- and embryo-specific Arabidopsis DLL promoter bears good potential for application in marker-free Cre/loxP self-excision strategy. Plant Cell Rep 34, 469–481 (2015). https://doi.org/10.1007/s00299-014-1726-0

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  • DOI: https://doi.org/10.1007/s00299-014-1726-0

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