Transgenic Research

, Volume 16, Issue 6, pp 771–781 | Cite as

pORE: a modular binary vector series suited for both monocot and dicot plant transformation

  • Catherine Coutu
  • James Brandle
  • Dan Brown
  • Kirk Brown
  • Brian Miki
  • John Simmonds
  • Dwayne D. Hegedus
Original Paper

Abstract

We present a series of 14 binary vectors suitable for Agrobacterium-mediated transformation of dicotyledonous plants and adaptable for biolistic transformation of monocotyledonous plants. The vector size has been minimized by eliminating all non-essential elements from the vector backbone and T-DNA regions while maintaining the ability to replicate independently. The smallest of the vector series is 6.3 kb and possesses an extensive multiple cloning site with 21 unique restriction endonuclease sites that are compatible with common cloning, protein expression, yeast two-hybrid and other binary vectors. The T-DNA region was engineered using a synthetic designer oligonucleotide resulting in an entirely modular system whereby any vector element can be independently exchanged. The high copy number ColE1 origin of replication has been included to enhance plasmid yield in Escherichia coli. FRT recombination sites flank the selectable marker cassette regions and allow for in planta excision by FLP recombinase. The pORE series consists of three basic types; an ‘open’ set for general plant transformation, a ‘reporter’ set for promoter analysis and an ‘expression’ set for constitutive expression of transgenes. The sets comprise various combinations of promoters (PHPL, PENTCUP2 and PTAPADH), selectable markers (nptII and pat) and reporter genes (gusA and smgfp).

Keywords

Agrobacterium tumefaciens Binary vector Plant transformation T-DNA Promoter Selectable marker 

Abbreviations

bp

Base pair

DNA

Deoxyribonucleic acid

dsDNA

Double stranded DNA

gusA

β-Glucuronidase

LB

Left T-DNA border

MCS

Multiple cloning site

nptII

Neomycin phosphotransferase II

OxOx

Oxalate oxidase

pat

Phosphinothricin acetyltransferase

PENTCUP2

Tobacco cryptic constitutive promoter

PCR

Polymerase chain reaction

PHPL

Arabidopsis thaliana hydroperoxide lyase promoter

PTAP

Triticum aestivum lipid transfer protein promoter

PTAPADH

Triticum aestivum lipid transfer protein promoter fused to an alcohol dehydrogenase intron

RB

Right T-DNA border

SDM

Site directed mutagenesis

smgfp

soluble modified green fluorescent protein

T-DNA

Transferred DNA

Ti

Tumour inducing

TNOS

Polyadenylation signal from the nopaline synthase gene

Vir

Virulence region of the Ti plasmid

References

  1. Azam N, Dixon JE, Sadowski PD (1997) Topological analysis of the role of homology in flp-mediated recombination. J Biol Chem 272:8731–8738PubMedCrossRefGoogle Scholar
  2. Bate NJ, Sivasankar S, Moxon C, Riley JM, Thompson JE, Rothstein SJ (1998) Molecular characterization of an Arabidopsis gene encoding hydroperoxide lyase, a cytochrome P-450 that is wound inducible. Plant Physiol 117:1393–1400PubMedCrossRefGoogle Scholar
  3. Bechtold N, Ellis J, Pelletier G (1993). In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thalianai plants. C R Acad Sci Paris Life Sci 316:1194–1199Google Scholar
  4. Clough S, Bent A (1998) Floral Dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743PubMedCrossRefGoogle Scholar
  5. Davis SJ, Vierstra RD (1998) Soluble, highly fluorescent variants of green fluorescent protein (gfp) for use in higher plants. Plant Mol Biol 36:521–528PubMedCrossRefGoogle Scholar
  6. Gelvin S (2000). Agrobacterium and plant genes involved in T-DNA transfer and integration. Ann Rev Plant Physiol Plant Mole Biol 51:223–256CrossRefGoogle Scholar
  7. Gelvin S (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67:16–37PubMedCrossRefGoogle Scholar
  8. Gidoni D, Bar M, Leshem B, Gilboa N, Mett A, Feiler J (2001) Embryonal recombination and germline inheritance of recombined Frt loci mediated by constitutively expressed Flp in tobacco. Euphyti J 121:145–156CrossRefGoogle Scholar
  9. Goderis IJ, De Bolle MF, Francois IE, Wouters PF, Broekaert WF, Cammue BP (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol Biol 50:17–27PubMedCrossRefGoogle Scholar
  10. Helens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832CrossRefGoogle Scholar
  11. Kim SR, Lee J, Jun SH, Park S, Kang HG, Kwon S, An G (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52:761–773PubMedCrossRefGoogle Scholar
  12. Luo H, Kausch AP (2002) Application of FLP/FRT site-specific DNA recombination system in plants. Genet Eng 24:1–16Google Scholar
  13. Luo H, Lyznik LA, Gidoni D, Hodges TK (2000) Flp-mediated recombination for use in hybrid plant production. Plant J 23:423–430PubMedCrossRefGoogle Scholar
  14. Malik K, Wu K, Li XQ, Martin-Heller T, Hu M, Foster E, Tian L, Wang C, Ward K, Jordan M, Brown D, Gleddie S, Simmonds D, Zheng S, Simmonds J, Miki B (2002) A constitutive gene expression system derived from the Tcup cryptic promoter elements. Theor Appl Genet 105:505–514PubMedCrossRefGoogle Scholar
  15. McCormac AC, Elliott MC, Chen DF (1997) pBecks: a plexible series of binary vectors for Agrobacterium-mediated plant transformation. Mol Biotechnol 8:199–213PubMedGoogle Scholar
  16. Nakamura Y, Gojobori T, Ikemura T (2000) Codon usage tabulated from the international DNA sequence databases: status for the Year 2000. Nucleic Acids Res 2000:292CrossRefGoogle Scholar
  17. Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual. Cold Spring Harbour Laboratory Press, New YorkGoogle Scholar
  18. Schafer U, Hegedus D, Bate NJ, Gleddie S, Brown DC (2002) Hydrogen peroxide lyase regulatory region. International Patent WO 2002/050291Google Scholar
  19. Sheng J, Citovsky V (1996) Agrobacterium-plant cell DNA transport: have virulence proteins, will travel. Plant Cell 8:1699–1710PubMedCrossRefGoogle Scholar
  20. Simmonds J, Leslie C, Harris L, Sharon A (2001) Wheat aleurone regulatory region. US Patent 6:326–528Google Scholar
  21. Wu K, Malik K, Tian L, Hu M, Martin T, Foster E, Brown D, Miki B (2001) Enhancers and core promoter elements are essential for the activity of a cryptic gene activation sequence from tobacco, Tcup. Mol Gen Genet 265:763–770Google Scholar
  22. Xiang C, Han P, Lutziger I, Wang K, Oliver DJ (1999) A mini binary vector series for plant transformation. Plant Mol Biol 40:711–717PubMedCrossRefGoogle Scholar
  23. Yenofsky R, Fine M, Pellew J (1990) A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc Natl Acad Sci USA 87:3435–3439PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Catherine Coutu
    • 1
  • James Brandle
    • 2
  • Dan Brown
    • 2
  • Kirk Brown
    • 2
  • Brian Miki
    • 3
  • John Simmonds
    • 3
  • Dwayne D. Hegedus
    • 1
  1. 1.Agriculture and Agrifood Canada, Saskatoon Research CentreSaskatoonCanada
  2. 2.Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research CenterLondonCanada
  3. 3.Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research CentreOttawaCanada

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