Advertisement

Molecular Breeding

, Volume 10, Issue 1–2, pp 31–44 | Cite as

Stable expression of Phytase (phyA) in canola (Brassica napus) seeds: towards a commercial product

  • Anne S. Ponstein
  • Jacob B. Bade
  • Theo C. Verwoerd
  • Lucy Molendijk
  • Joost Storms
  • Rob F. Beudeker
  • Jan Pen
Article

Abstract

TheAspergillus niger gene encoding phytase(phyA) was expressed in canola (Brassicanapus). Phytase expression is controlled by the seed-specificcruciferin (CruA) promoter. Secretion of the enzyme was aimed for byincorporating the cruciferin signal peptide in the expression construct.Transgenic canola lines were generated by Agrobacteriummediated transformation using nptII as the selectable marker. Ninety-fiveindependent transgenic events were generated. Phytase expression in the T1seedsranged from 0 to 600 U/g seed. Single-copy lines were selected(based on segregation for kanamycin resistance, phytase expression and Southernanalyses) from originally multi-copy transgenic lines. Phytase was expressed inthese sub-lines up to 103 U/g. Expression levels were monitoredthrough an additional 3–4 generations (in the greenhouse and in thefield)and the accumulation of phytase appeared to be fairly stable. In the expressionrange studied, phytase expression was gene-dosage dependent.

Brassica napus Cruciferin promoter Phytase Seed-specific expression 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Austin S., Bingham E.T., Koegel R.G., Mathews D.E., Shahan M.N., Straub R.J. et al. 1994. An overview of a feasibility study for the production of industrial enzymes in transgenic alfalfa. Recombinant DNA Technology II: 234-244.Google Scholar
  2. Bevan M. 1984. Binary Agrobacterium vectors for plant transformation. Nucl. Acid Res. 12: 8711-8721.Google Scholar
  3. Blundy K.S., Blundy M.A. and Crouch L. 1991. Differential expression of members of the napin storage protein gene family during embryogenesis in Brassica napus. Plant Mol. Biol. 17: 1099-1104.PubMedGoogle Scholar
  4. Bradford M.M. 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.CrossRefPubMedGoogle Scholar
  5. Brinch-Pedersen H., Olesen A., Rasmussen S.K. and Holm P.B. 2000. Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase. Molecular Breeding 6: 195-206.Google Scholar
  6. Chang M.T., Keeling P.L. and Wilhelm J.C. 1999. Animal Feed with Low Phytic Acid, Oil Burdened and Protein Ladengrain. WO Patent 99/02668.Google Scholar
  7. DeLisle A.J. and Crouch M.L. 1989. Seed storage protein transcription and mRNA levels in Brassica napus during development and in response to exogenous abscisic acid. Plant Physiol. 91: 617-623.Google Scholar
  8. Denbow D.M., Grabau E.A., Lacy G.H., Kornegay E.T., Russell D.R. and Umbeck P.F. 1998. Soybeans transformed with a fungal phytase gene improve phosphorus availability for broilers. Poult. Sci. 77: 878-881.PubMedGoogle Scholar
  9. DeSilva J., Robinson S.J. and Afford R. 1992. The isolation and functional characterisation of a Brassica napus acyl carrier protein 5' flanking region involved in the regulation of seed storage lipid synthesis. Plant Mol. Biol. 18: 1163-1172.PubMedGoogle Scholar
  10. Ditta G., Stanfield G., Corbiu D. and Helinski D. 1980. Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77: 7347-7351.PubMedGoogle Scholar
  11. Gamborg O.L., Miller R.A. and Ojima K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151-158.PubMedGoogle Scholar
  12. Gibson D.M. and Ullah A.B.J. 1990. Phytase and their action on phytic acid. In: Morré D.J., Boss W.F. and Loewus F.A. (eds), Inositol Metabolism in Plants. Wiley-Liss, Chichester, pp. 77-92.Google Scholar
  13. Hood E.E., Gelvin S.B., Melchers L.S. and Hoekema A. 1993. New Agrobacterium helper plasmids for gene transfer to plants. Trans. Res. 2: 208-218.Google Scholar
  14. Horsch R.B., Fry J.E., Hoffmann N.L., Eichholtz D., Rogers S.G. and Fraley R.T. 1985. A simple and general method for transferring genes into plants. Science 227: 1229-1231.Google Scholar
  15. Josefsson L.G., Lenman M., Ericson M.L. and Rask L. 1987. Structure of a gene encoding the 1.7 S storage protein, napin, from Brassica napus. J. Biol. Chem. 262: 12196-12201.PubMedGoogle Scholar
  16. Kohno-Murase J., Murase M., Ichikawa H. and Imamura J. 1995. Improvement in the quality of seed storage protein by transformation of Brassica napus with an antisense gene for cruciferin. Theor. Appl. Genet. 91: 627-631.Google Scholar
  17. Lassner M.W., Peterson P. and Yoder J.I. 1989. Simultaneous amplification of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their progeny. Plant Mol. Biol. Rep. 7: 116-128.Google Scholar
  18. Li J., Hegeman C.E., Hanlon R.W., Lacy G.H., Denbow D.M. and Grabau E.A. 1997. Secretion of active recombinant phytase from soybean cell-suspension cultures. Plant Physiol. 114: 1103-1111.PubMedGoogle Scholar
  19. Lolas M. and Markakis P. 1977. Phytase of navy beans. J. Food Sci. 42: 1094-1097.Google Scholar
  20. Lott J.N.A. 1984. Accumulation of seed reserves of phosphorus and other minerals. Seed Physiology 1: 139-166.Google Scholar
  21. Martino-Catt S.J., Wang H., Beach L.R., Bowen B.A. and Wang X. 1999. Genes Controlling Phytate Metabolism in Plants and Uses Thereof. WO patent 99/05298.Google Scholar
  22. Moloney M.M., Walker J.M. and Sharma K.K. 1989. High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep. 8: 238-242.Google Scholar
  23. Murashige T. and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-479.Google Scholar
  24. Nelson T.S., Shieh T.R., Wodzinski R.J. and Ware J.H. 1971. Effect of supplemental phytase on the utilization of phytate phophorus by chicks. J. Nutr. 101: 1289-1294.PubMedGoogle Scholar
  25. Pen J., Verwoerd T.C., Van Paridon P.A., Beudeker R.F., Van den Elzen P.J.M., Geerse K. et al. 1993. Phytase-containing transgenic seeds as a novel feed additive for improved phosphorus utilization. Bio/Technology 11: 811-814.Google Scholar
  26. Raboy V. 1997. Low phytic acid mutants and selection thereof. US patent 5,689,504.Google Scholar
  27. Ryan A.J., Royal C.L., Hutchinson J. and Shaw C.H. 1989. Genomic sequence of a 12S seed storage protein from oilseed rape (Brassica napus c.v. Jet Neuf). Nucl. Acids Res. 17: 3584.PubMedGoogle Scholar
  28. Sambrook J., Fritsch E.F. and Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.Google Scholar
  29. Sijmons P.C.M., Dekker B.M.M., Schrammeijer B., Verwoerd T.C., Van den Elzen P.J.M. and Hoekema A. 1990. Production of correctly processed human serum albumin in transgenic plants. Bio/Technology 8: 217-221.CrossRefPubMedGoogle Scholar
  30. Simons P.C.M., Versteegh H.A.J., Jongbloed A.W., Kemme P.A., Slump P., Bos K.D. et al. 1990. Improved phosphorus availability by microbial phytase in broilers and pigs. Brit. J. Nutr. 64: 525-540.PubMedGoogle Scholar
  31. Sjödahl S., Gustavsson H.O., Rödin J., Lenman M., Hoglund A.S. and Rask L. 1993. Cruciferin gene families are expressed coordinately but with tissue-specific differences during Brassica napus seed development. Plant Mol. Biol. 23: 1165-1176.PubMedGoogle Scholar
  32. Sjödahl S., Gustavsson H.O., Rödin J. and Rask L. 1995. Deletion analysis of the Brassica napus cruciferin gene cru 1 promoter in transformed tobacco: promoter activity during early and late stages of embryogenesis is influenced by cis-acting elements in partially separate regions. Planta 197: 264-271.PubMedGoogle Scholar
  33. Van Hartingsveldt W., Van Zijl C.M.J., Harteveld G.M., Gouka R.J., Suykerbuyk M.E.G., Luiten R.G.M. et al. 1993. Cloning, characterization and overexpression of the phytase-encoding gene (phyA) of Aspergillus niger. Gene 127: 87-94.PubMedGoogle Scholar
  34. Verwoerd T.C., Van Paridon P.A., Van Ooyen A.J.J., Van Lent J.W.M., Hoekema A. and Pen J. 1995. Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves. Plant Physiol. 109: 1199-1205.PubMedGoogle Scholar
  35. Wodzinski R.J. and Ullah A.H.J. 1996. Phytase. Advances in Appli. Microbiol., pp. 263-302.Google Scholar
  36. Yenofsky R.L., Fine M. and Pellow J.W. 1990. A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc. Natl. Acad. Sci. USA 87: 3435-3439.PubMedGoogle Scholar
  37. Zhang Z.B., Kornegay E.T., Radcliffe J.S., Denbow D.M., Veit H.P. and Larsen C.T. 2000a. Comparison of genetically engineered microbial and plant phytase for young broilers. Poult. Sci. 79: 709-717.PubMedGoogle Scholar
  38. Zhang Z.B., Kornegay E.T., Radcliffe J.S., Denbow D.M. and Veit H.P. 2000b. Comparison of phytase from genetically engineered Aspergillus and canola in weanling pig diets. J. Anim. Sci. 78: 2868-2878.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Anne S. Ponstein
    • 1
  • Jacob B. Bade
    • 1
  • Theo C. Verwoerd
    • 1
  • Lucy Molendijk
    • 1
  • Joost Storms
    • 1
  • Rob F. Beudeker
    • 2
  • Jan Pen
    • 3
  1. 1.Syngenta MogenLeidenThe Netherlands
  2. 2.DelftThe Netherlands
  3. 3.PlantZymeLeidenThe Netherlands

Personalised recommendations