Molecular Breeding

, Volume 19, Issue 3, pp 241–253 | Cite as

Cloning of new rubisco promoters from Brassica rapa and determination of their activity in stably transformed Brassica napus and Nicotiana tabacum plants

  • Andrey Anisimov
  • Kimmo Koivu
  • Anne Kanerva
  • Seppo Kaijalainen
  • Kari Juntunen
  • Viktor Kuvshinov
Original Paper


The aim of our study was to identify the highest expressing rubisco small subunit (RbcS) promoters (pRbcS) from the cotyledons of germinating seedlings of Brassica rapa var. oleifera to drive high-level and preferably stage-specific transgenic protein expression in Brassicaceae plants. We cloned four new pRbcS promoters using several approaches, including the construction of a cDNA library and use of genome walking technique. Real-time PCR analysis of RbcS mRNA expression clearly showed that two of these promoters exhibited the highest activity on the germination stage of plant development. We used gusA expression as a reporter of promoter activity in Brassica napus and Nicotiana tabacum plants that were transformed with the constructs using an Agrobacterium-mediated transformation strategy. The mRNA level of RbcS and of gusA was quantified in transformed plants. The data obtained demonstrate that the promoter most active in seedlings under native conditions was also most active in transgenic constructs at the same stage of plant development. The fine structure of the promoters is discussed herein.


Brassica species and Nicotiana tabacum transformations Rubisco promoters Rubisco small subunit 



β-d-glucuronidase, encoded by the gusA gene


Promoter of RbcS gene


Rubisco small subunit



We are grateful to Dr. Nick Domansky and Ghebre Sebahtu for carrying out the Northern analysis, and Tatiana Shavkera and Svetlana Kuvshinova for their assistance in the transformation and in vitro culture work. We also thank Maritta Timonen and Dr. Eric Murphy for their generous help in preparation of the manuscript and two anonymous reviewers for their helpful comments. This Work was funded in part by the National Technology Agency of Finland (TEKES).


  1. Anissimov A, Kaijalainen S, Koivu K et al. (2004) Novel rubisco promoters and uses thereof. US patent application 20050241022, 2 July 2004Google Scholar
  2. Arguello-Astorga G, Herrera-Estrella L (1998) Evolution of light-regulated plant promoters. Annu Rev Plant Physiol Plant Mol Biol 49:525–555PubMedCrossRefGoogle Scholar
  3. Avesani L, Falorni A, Tornielli GB et al. (2003) Improved in planta expression of the human islet autoantigen glutamic acid decarboxylase (GAD65). Transgenic Res 12:203–212PubMedCrossRefGoogle Scholar
  4. Bai Y, Nikolov ZL, Glatz CE (2002) Aqueous extraction of beta-glucuronidase from transgenic canola: kinetics and microstructure. Biotechnol Prog 18:1301–1305PubMedCrossRefGoogle Scholar
  5. Beck I, Wittig C, Link G (1995) Transient expression activity of RbcS promoter regions from Brassica napus in mesophyll protoplasts from Nicotiana tabacum. Bot Acta 108:327–333Google Scholar
  6. Carrasco P, Manzara T, Gruissem W (1993) Developmental and organ-specific changes in DNA-protein interactions in the tomato rbcS3B and rbcS3C promoter regions. Plant Mol Biol 21:1–15PubMedCrossRefGoogle Scholar
  7. Charity JA, Anderson MA, Bittisnich DJ et al. (1999) Transgenic tobacco and peas expressing a proteinase inhibitor from Nicotiana alata have increased insect resistance. Mol Breed 5:357–365CrossRefGoogle Scholar
  8. De Almeida ERP, Gossele V, Muller CG et al. (1989) Transgenic expression of two marker genes under the control of an Arabidopsis rbcS promoter: Sequences encoding the Rubisco transit peptide increase expression levels. Mol Genet Genomics 218:78–86CrossRefGoogle Scholar
  9. Dean C, Pichersky E, Dunsmuir P (1989) Structure, evolution, and regulation of RbcS genes in higher plants. Annu Rev Plant Physiol Plant Mol Biol 40:415–439CrossRefGoogle Scholar
  10. 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 19:71–74PubMedCrossRefGoogle Scholar
  11. Dedonder A, Rethy R, Fredericq H et al. (1993) Arabidopsis rbcS genes are differentially regulated by light. Plant Physiol 101:801–808PubMedCrossRefGoogle Scholar
  12. Fiebig C, Link G (1992) 5’-Upstream cis-elements and binding factor(s) potentially involved in light-regulated expression of a Brassica napus rbcS gene. Curr Genet 21:161–168PubMedCrossRefGoogle Scholar
  13. Fiebig C, Kretzschmar F, Sprenger I, Link G (1990a) Sequence characteristics and transcripts of rbcS genes from Brassica napus: temporal and spatial expression during crucifer seedling morphogenesis. Bot Acta 103:258–265Google Scholar
  14. Fiebig C, Neuhaus H, Teichert J et al. (1990b) Temporal and spatial pattern of plastid gene expression during crucifer seedling development and embryogenesis. Planta 181:191–198CrossRefGoogle Scholar
  15. Fukuda Y, Nishikawa S (2003) Matrix attachment regions enhance transcription of a downstream transgene and the accessibility of its promoter region to micrococcal nuclease. Plant Mol Biol 51:665–675PubMedCrossRefGoogle Scholar
  16. Galili S, Avivi Y, Millet E, Feldman M (2000) RFLP-based analysis of three RbcS subfamilies in diploid and polyploid species of wheat. Mol Gen Genet 263:674–680PubMedCrossRefGoogle Scholar
  17. Gyulai G, Kiss E, Heszky LE (1992) Biotechnology of rapeseed (Brassica napus L.). Acta Agron Hung 41:277–287Google Scholar
  18. Hou BK, Zhou YH, Wan LH et al. (2003) Chloroplast transformation in oilseed rape. Transgenic Res 12:111–114PubMedCrossRefGoogle Scholar
  19. Jang IC, Nahm BH, Kim JK (1999) Subcellular targeting of green fluorescent protein to plastids in transgenic rice plants provides a high-level expression system. Mol Breed 5:453–461CrossRefGoogle Scholar
  20. Joly S, Rauscher JT, Sherman-Broyles SL et al. (2004). Evolutionary dynamics and preferential expression of homeologous 18S-5.8S-26S nuclear ribosomal genes in natural and artificial glycine allopolyploids. Mol Biol Evol 21:1409–1421PubMedCrossRefGoogle Scholar
  21. Khoudi H, Laberge S, Ferullo JM et al. (1999) Production of a diagnostic monoclonal antibody in perennial alfalfa plants. Biotechnol Bioeng 64:135–143PubMedCrossRefGoogle Scholar
  22. Kim UJ, Shizuya H, de Jong PJ et al. (1992) Stable propagation of cosmid sized human DNA inserts in an F factor based vector. Nucleic Acids Res 20:1083–1085PubMedCrossRefGoogle Scholar
  23. Koivu K (2004) Novel sprouting technology for recombinant protein production. In: Fischer R, Schillberg S (eds) Molecular farming. Wiley-VCH Verlag, WeinheimGoogle Scholar
  24. Krebbers E, Seurinck J, Herdies L et al. (1988) Four genes in two diverged subfamilies encode the rubulose-1, 5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana. Plant Mol Biol 11:745–759CrossRefGoogle Scholar
  25. Kuvshinov V, Koivu K, Kanerva A, Pehu E (2001) Transgenic crop plants expressing synthetic cry9Aa gene are protected against insect damage. Plant Science 160:341–353PubMedCrossRefGoogle Scholar
  26. Kuvshinov V, Anissimov A, Yahya BM (2004) Barnase gene inserted in the intron of GUS – a model for controlling transgene flow in host plants. Plant Sci 167:173–182CrossRefGoogle Scholar
  27. Kvarnheden A, Tandre K, Engstrom P (1995) A cdc2 homologue and closely related processed retropseudogenes from Norway spruce. Plant Mol Biol 27:391–403PubMedCrossRefGoogle Scholar
  28. Mankin SL, Allen GC, Phelan T, Spiker S, Thompson WF (2003) Elevation of transgene expression level by flanking matrix attachment regions (MAR) is promoter dependent: a study of the interactions of six promoters with the RB7 3’MAR. Transgenic Res 12:3–12PubMedCrossRefGoogle Scholar
  29. Manzara T, Carrasco P, Gruissem W (1991) Developmental and organ-specific changes in promoter DNA-protein interactions in the tomato rbcS gene family. Plant Cell 3:1305–1316PubMedCrossRefGoogle Scholar
  30. Manzara T, Carrasco P, Gruissem W (1993) Developmental and organ-specific changes in DNA-protein interactions in the tomato rbcS1, rbcS2 and rbcS3A promoter regions. Plant Mol Biol 21:69–88PubMedCrossRefGoogle Scholar
  31. McGrath JM, Jancso MM, Pichersky E (1993) Duplicate sequences with a similarity to expressed genes in the genome of Arabidopsis thaliana. Theor Appl Genet 86:880–888CrossRefGoogle Scholar
  32. Meier I, Callan KL, Fleming AJ, Gruissem W (1995) Organ-specific differential regulation of a promoter subfamily for the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit genes in tomato. Plant Physiol 107:1105–1118 PubMedCrossRefGoogle Scholar
  33. Nantel AM, Lafleur F, Boivin R et al. (1991) Promoter for a Brassica napus ribulose bisphosphate carboxylase/oxygenase small subunit gene binds multiple nuclear factors and contains a negative-strand open reading frame encoding a putative transmembrane protein. Plant Mol Biol 16:955–966PubMedCrossRefGoogle Scholar
  34. Niwa Y, Goto K, Shimizu M, Kobayashi H (1997) Chromosomal mapping of genes in the RBCS family in Arabidopsis thaliana. DNA Res 4:341–343PubMedCrossRefGoogle Scholar
  35. Outchkourov NS, Peters J, de Jong J et al. (2003a) The promoter-terminator of chrysanthemum rbcS1 directs very high expression levels in plants. Planta 216:1003–1012Google Scholar
  36. Outchkourov NS, Rogelj B, Strukelj B, Jongsma MA (2003b) Expression of sea anemone equistatin in potato. Effects of plant proteases on heterologous protein production. Plant Physiol 133:379–390CrossRefGoogle Scholar
  37. Park Y, Cheong H (2002) Expression and production of recombinant human interleukin-2 in potato plants. Protein Expr Purif 25:160–165PubMedCrossRefGoogle Scholar
  38. Peeters K, De Wilde C, Depicker A (2001) Highly efficient targeting and accumulation of a F(ab) fragment within the secretory pathway and apoplast of Arabidopsis thaliana. Eur J Biochem 268:4251–4260PubMedCrossRefGoogle Scholar
  39. PlantCARE: plant cis-acting regulatory elements.
  40. PLACE: A database of plant cis-acting regulatory DNA elements.
  41. Post-Beittenmiller MA, Schmid KM, Ohlrogge JB (1989) Expression of holo and apo forms of spinach acyl carrier protein-I in leaves of transgenic tobacco plants. Plant Cell 1:889–899PubMedCrossRefGoogle Scholar
  42. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, PlainviewGoogle Scholar
  43. Sasanuma T (2001) Characterization of the rbcS multigene family in wheat: subfamily classification, determination of chromosomal location and evolutionary analysis. Mol Genet Genomics 265:161–171PubMedCrossRefGoogle Scholar
  44. Sasanuma T, Miyashita NT (1998) Subfamily divergence in the multigene family of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS) in Triticeae and its relatives. Genes Genet Syst 73:297–309PubMedCrossRefGoogle Scholar
  45. Schillberg S, Fischer R, Emans N (2003) Molecular farming of recombinant antibodies in plants. Cell Mol Life Sci 60:433–445PubMedCrossRefGoogle Scholar
  46. Simillion C, Vandepoele K, Van Montagu MCE et al. (2002) The hidden duplication past of Arabidopsis thaliana. Proc Natl Acad Sci USA 99:13627–13632PubMedCrossRefGoogle Scholar
  47. Singh MB, Bhalla PL (2006) Recombinant expression systems for allergen vaccines. Inflamm Allergy Drug Targets 5:53–59PubMedGoogle Scholar
  48. Smith ML, Keegan ME, Mason HS, Shuler ML (2002) Factors important in the extraction, stability and in vitro assembly of the hepatitis B surface antigen derived from recombinant plant systems. Biotechnol Prog 18:538–550PubMedCrossRefGoogle Scholar
  49. Snowdon RJ, Friedrich T, Friedt W, Köhler W (2002) Identifying the chromosomes of the A- and C-genome diploid Brassica species B. rapa (syn. campestris) and B. oleracea in their amphidiploid B. napus. Theor Appl Genet 104:533–538PubMedCrossRefGoogle Scholar
  50. Song K, Lu P, Tang K, Osborn TC (1995) Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution. Proc Natl Acad Sci USA 92:7719–7723PubMedCrossRefGoogle Scholar
  51. Spreitzer RJ (2003) Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 414:141–149PubMedCrossRefGoogle Scholar
  52. Spreitzer RJ, Salvucci ME (2002) Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Annu Rev Plant Biol 53:449–475PubMedCrossRefGoogle Scholar
  53. Stoger E, Vaquero C, Torres E et al. (2000) Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol 42:583–590PubMedCrossRefGoogle Scholar
  54. Sugita M, Manzara T, Pichersky E et al. (1987) Genomic organization, sequence analysis and expression of all five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato. Mol Gen Genet 209:247–256PubMedCrossRefGoogle Scholar
  55. Vaquero C, Sack M, Schuster F et al. (2002) A carcinoembryonic antigen-specific diabody produced in tobacco. FASEB J 16:408–410PubMedGoogle Scholar
  56. Wolter FP, Fritz CC. Willmitzer L et al. (1988) rbcS genes in Solanum tuberosum: conservation of transit peptide and exon shuffling during evolution. Proc Natl Acad Sci USA 85:846–850PubMedCrossRefGoogle Scholar
  57. Zambryski P, Joos H, Genetello C et al. (1983) Ti-plasmid vector for the introduction of DNA into plant-cells without alteration of their normal regeneration capacity. EMBO J 2:2143–2150PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Andrey Anisimov
    • 1
  • Kimmo Koivu
    • 1
  • Anne Kanerva
    • 1
  • Seppo Kaijalainen
    • 1
  • Kari Juntunen
    • 2
  • Viktor Kuvshinov
    • 1
  1. 1.Helsinki Business & Science ParkUniCrop Ltd.HelsinkiFinland
  2. 2.Department of Biosciences, Division of BiochemistryUniversity of HelsinkiHelsinkiFinland

Personalised recommendations