Skip to main content

Advertisement

SpringerLink
Log in

Tobacco ribosomal DNA spacer element elevates Bowman–Birk inhibitor expression in tomato plants

  • Genetic Transformation and Hybridization
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Amplification promoting sequence (aps), from tobacco rDNA, was found to induce amplification and enhances the expression of heterologous genes, consequently increasing the expression of transgenic proteins in tobacco. In this report we demonstrate that aps element also affects integration, transcription, and translation of a soybean protease inhibitor, Bowman–Birk inhibitor (BBI), in transgenic tomato plants and quantifies its effects in different expression vectors. A synthetic bbi gene was constructed, based on the wild-type gene containing two independent inhibition sites; trypsin and chymotrypsin. Transformation vectors were designed using two different promoters; the tomato fruit specific E8 promoter and the constitutively active 35S CaMV promoter. These vectors were transformed into ‘Moneymaker’ tomato plants. In tomato fruits and leaves, aps caused a 3-fold increase in bbi mRNA levels when compared to the lines without aps. Similar increases were obtained in plants expressing bbi controlled by E8 or 35S CaMV promoters. Also, the level of BBI protein expression in aps-transformed plants was 3 fold-higher than in plants without aps. This is the first report of aps effect on the enhanced gene expression and transgenic protein production in plant other than tobacco.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Beekwilder J, Schipper B, Bakker P, Bosch D, Jongsma M (2000) Characterization of potato proteinase inhibitor II reactive site mutants. Eur J Biochem 267:1975–1984

    Article  PubMed  Google Scholar 

  • Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721

    PubMed  Google Scholar 

  • Billings PC, St Clair WH, Maki PA, Kennedy AR (1992) Distribution of the Bowman Birk protease inhibitor in mice following oral administration. Cancer Lett 62:191–197

    Article  PubMed  Google Scholar 

  • Birk Y (1985) The Bowman–Birk inhibitor. Trypsin- and chymotrypsin-inhibitor from soybeans. Int J Pept Protein Res 25:113–131

    PubMed  Google Scholar 

  • Bogorad L (2000) Engineering chloroplasts: an alternative site for foreign genes, proteins, reactions and products. Trends Biotechnol 18:257–263

    Google Scholar 

  • Borisjuk NV, Davidjuk YM, Kostishin SS, Miroshnichenco GP, Velasco R, Hemleben V, Volkov RA (1997) Structural analysis of rDNA in the genus Nicotiana. Plant Mol Biol 35:655–660

    Article  PubMed  Google Scholar 

  • Borisjuk N, Sitailo L, Adler K, Malysheva L, Tewes A, Borisjuk L, Manteuffel R (1998) Calreticulin expression in plant cells: developmental regulation, tissue specificity and intracellular distribution. Planta 206:504–514

    Article  PubMed  Google Scholar 

  • Borisjuk N, Borisjuk L, Komarnytsky S, Timeva S, Hemleben V, Gleba Y, Raskin I (2000) Tobacco ribosomal DNA spacer element stimulates amplification and expression of heterologous genes. Nat Biotechnol 18:1303–1306

    Article  PubMed  Google Scholar 

  • Chavan JK, Hejgaard J (1981) Detection and partial characterization of subtilism inhibitors in Legume seeds by isoelectric focusing. J Sci Food Agric 32:857–862

    PubMed  Google Scholar 

  • Deikman J, Fischer RL (1988) Interaction of a DNA binding factor with the 5′-flanking region of an ethylene-responsive fruit ripening gene from tomato. EMBO J 7:3315–3320

    PubMed  Google Scholar 

  • Dinant S, Ripoll C, Pieper M, David C (2004) Phloem specific expression driven by wheat dwarf geminivirus V-sense promoter in transgenic dicotyledonous species. Physiol Plant 121:108–116

    Article  PubMed  Google Scholar 

  • Dittmann K, Knaus-Dittmann D, Mayer C, Rodemann HP (2001) Bowman–Birk proteinase inhibitor-mediated radioprotection against UV irradiation is TP53-dependent and associated with stimulation of nucleotide excision repair. Radiat Environ Biophys 40:163–167

    Article  PubMed  Google Scholar 

  • Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7:152–158

    Article  PubMed  Google Scholar 

  • Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18:1151–1155

    Article  PubMed  Google Scholar 

  • Hemann C, Gartner E, Weidle UH, Grummt F (1994) High-copy expression vector based on amplification-promoting sequences. DNA Cell Biol 13:437–445

    PubMed  Google Scholar 

  • Hetzroni A (1997) The protease inhibitor Bowman–Birk-Inhibitor (BBI): expression in the bacteria E. coli and the yeast Pichia pastoris, isolating, characterization and studying the relation between structure and biological activity. Biochemistry, Hebrew University, Jerusalem, Israel

    Google Scholar 

  • Hood EE, Woodard SL, Horn ME (2002) Monoclonal antibody manufacturing in transgenic plants—myth and realities. Curr Opin Biotechnol 13:630–635

    Article  PubMed  Google Scholar 

  • Jani D, Meena LS, Rizwan-ul-Haq QM, Singh Y, Sharma AK, Tyagi AK (2002) Expression of cholera toxin B subunit in transgenic tomato plants. Transgenic Res 11:447–454

    Article  PubMed  Google Scholar 

  • Jering H, Tschesche H (1976) Replacement of lysine by arginine, phenylalanine and tryptophan in the reactive site of the bovine trypsin-kallikrein inhibitor (Kunitz) and change of the inhibitory properties. Eur J Biochem 61:453–463

    Article  PubMed  Google Scholar 

  • Kennedy AR (1998) The Bowman–Birk inhibitor from soybeans as an anticarcinogenic agent. Am J Clin Nutr 68:1406S–1412S

    PubMed  Google Scholar 

  • Komarnytsky S, Gaume A, Garvey A, Borisjuk N, Raskin I (2004) A quick and efficient system for antibiotic-free expression of heterologous genes in tobacco roots. Plant Cell Rep 22:765–773

    Article  PubMed  Google Scholar 

  • Kusnadi AR, Hood EE, Witcher DR, Howard JA, Nikolov ZL (1998) Production and purification of two recombinant proteins from transgenic corn. Biotechnol Prog 14:149–155

    Article  PubMed  Google Scholar 

  • Larrick JW, Thomas DW (2001) Producing proteins in transgenic plants and animals. Curr Opin Biotechnol 12:411–418

    Article  PubMed  Google Scholar 

  • Levin JS, Thompson WF, Csinos AS, Stephenson MG, Weissinger AK (2005) Matrix attachment regions increase the efficiency and stability of RNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco. Transgenic Res 14:193–206

    Article  PubMed  Google Scholar 

  • McGarvey PB, Hammond J, Dienelt MM, Hooper DC, Fu ZF, Dietzschold B, Koprowski H, Michaels FH (1995) Expression of the rabies virus glycoprotein in transgenic tomatoes. Biotechnology 13:1484–1487

    Article  PubMed  Google Scholar 

  • Mehta RA, Cassol T, Li N, Ali N, Handa AK, Mattoo AK (2002) Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality, and vine life. Nat Biotechnol 20:613–618

    Article  PubMed  Google Scholar 

  • Pfitzner AJ (1998) Transformation of tomato. Methods Mol Biol 81:359–363

    PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis TA (1989) Molecular cloning: A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Sandhu JS, Krasnyanski SF, Domier LL, Korban SS, Osadjan MD, Buetow DE (2000) Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F protein induces a systemic immune response. Transgenic Res 9:127–135

    Article  PubMed  Google Scholar 

  • Sawada K, Hasegawa M, Tokuda L, Kameyama J, Kodama O, Kohchi T, Yoshida K, Shinmyo A (2004) Enhanced resistance to blast fungus and bacterial blight in transgenic rice constitutively expressing OsSBP, a Rice homologue of mammalian selenium-binding proteins. Biosci Biotechnol Biochem 68:873–880

    Google Scholar 

  • Shure M, Wessler S, Fedoroff N (1983) Molecular identification and isolation of the Waxy locus in maize. Cell 35:225–233

    Article  PubMed  Google Scholar 

  • Stoger E, Sack M, Fischer R, Christou P (2002) Plantibodies: applications, advantages and bottlenecks. Curr Opin Biotechnol 13:161–166

    Article  PubMed  Google Scholar 

  • Sung-Ryul K, Jinwon L, Sung-Hoon J, Sunhee P, Hong-Gyu K, Soontae K, Gynheung A (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52:761–773

    Article  PubMed  Google Scholar 

  • Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM, Arntzen CJ (1998) Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat Med 4:607–609

    Article  PubMed  Google Scholar 

  • Williamson VM (1998) Root-knot nematode resistance genes in tomato and their potential for future use. Annu Rev Phytopathol 36:277–293

    Article  PubMed  Google Scholar 

  • Woodard SL, Mayor JM, Bailey MR, Barker DK, Love RT, Lane JR, Delaney DE, McComas-Wagner JM, Mallubhotla HD, Hood EE, Dangott LJ, Tichy SE, Howard JA (2003) Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants. Biotechnol Appl Biochem 38:123–130

    Article  PubMed  Google Scholar 

  • Yakoby N, Raskin I (2004) A simple method to determine trypsin and chymotrypsin inhibitory activity. J Biochem Biophys Methods 59:241–251

    Article  PubMed  Google Scholar 

  • Yu J, Langridge W (2003) Expression of rotavirus capsid protein VP6 in transgenic potato and its oral immunogenicity in mice. Transgenic Res 12:163–169

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We are thankful to Robert Fischer for providing us the E8 promoter construct. We greatly appreciate the experience and advice of David Kobiler during the antibody preparation. We thank Christophe Ripoll, Slavko Komarnytsky, and Nikolai Borisjuk for the fruitful discussions and critical remarks. Also, we are grateful to Ivan Jenkins and Reni Pouleva for their technical support. This research was supported by Vaadia-BARD Postdoctoral Award No. FI-302-2000 from BARD, The United States – Israel Binational Agricultural Research and Development Fund (granted to N.Y.), by Phytomedics Inc., Rutgers, The State University of New Jersey, and NJ Agricultural Experiment Station

Author information

Author notes

  1. Nir Yakoby

    Present address: Carl Icahn Laboratory, Lewis–Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, 08544, USA

Authors and Affiliations

  1. Biotech Center, Cook College, Rutgers University, Foran Hall, 59 Dudley Road, New Brunswick, NJ, 08901-8520, USA

    Nir Yakoby, Alison Garvey & Ilya Raskin

Authors
  1. Nir Yakoby
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alison Garvey
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ilya Raskin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nir Yakoby.

Additional information

Communicated by L. Peña

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yakoby, N., Garvey, A. & Raskin, I. Tobacco ribosomal DNA spacer element elevates Bowman–Birk inhibitor expression in tomato plants. Plant Cell Rep 25, 573–581 (2006). https://doi.org/10.1007/s00299-005-0101-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-005-0101-6

Key words

Search

Navigation