Plant Cell Reports

, 25:124 | Cite as

Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola

  • Ri-He Peng
  • Quan-Hong Yao
  • Ai-Sheng Xiong
  • Zong-Ming Cheng
  • Yi Li
Genetic Transformation and Hybridization


Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems.


Agrobacterium-mediated transformation Aspergillus phytase Brassica napus Codon modification Endoplasmic reticulum retention signal 





modified phytase gene


pathogen-related protein S


scaffold attachment regions



This research was supported by the Science Commission of Shanghai, project number 993913002. We thank Lori Osburn for proof-reading the manuscript.


  1. Allen GC, Hall G Jr, Michalowski S, Newman W, Spiker S, Weissinger AK, Thompson WF (1996) High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco. Plant Cell 8:899–913CrossRefPubMedGoogle Scholar
  2. Akashi H (2001) Gene expression and molecular evolution. Curr Opin Genet Dev 11:660–666CrossRefPubMedGoogle Scholar
  3. Austin S, Bingham ET, Koegel RG, Mathews DE, Shahan MN, Straub RJ, Burgess RR (1994) An overview of a feasibility study for the production of industrial enzymes in transgenic alfalfa. Ann NY Acad Sci 721:234–244PubMedCrossRefGoogle Scholar
  4. 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 72:248–254CrossRefPubMedGoogle Scholar
  5. Brinch-Pedersen H, Hatzack F, Sorensen LD, Holm PB (2003) Concerted action of endogenous and heterologous phytase on phytic acid degradation in seed of transgenic wheat (Triticum aestivum L.). Transgenic Res 12:649–659CrossRefPubMedGoogle Scholar
  6. Brinch-Pedersen H, Sorensen LD, Holm PB (2002) Engineering crop plants: getting a handle on phosphate. Trends Plant Sci 7:118–125CrossRefPubMedGoogle Scholar
  7. Campbell WH, Gowi G (1990) Codon usage in higher plants, green algae, and cyanobacteria. Plant Physiol 92:1–11PubMedCrossRefGoogle Scholar
  8. Cheryan M (1980) Phytic acid interactions in food systems. Crit Rev Food Sci Nutr 13:297–335PubMedCrossRefGoogle Scholar
  9. Conrad U, Fiedler U (1998) Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol Biol 38:101–109CrossRefPubMedGoogle Scholar
  10. Cornelissen BJC, Hooft van Huijsduijnen RAM, Bol JF (1986) A tobacco mosaic virus-induced tobacco protein is homologous to the sweet-tasting protein thaumatin. Nature 321:531–532CrossRefPubMedGoogle Scholar
  11. Denbow DM, Grabau EA, Lacy GH, Kornegay ET, Russell DR, Umbeck PF (1998) Soybeans transformed with a fungal phytase gene improve phosphorus availability for broilers. Poult Sci 77:878–881PubMedGoogle Scholar
  12. Dvorakova J (1998) Phytase: sources, preparation and exploitation. Folia Microbiol 43:323–338CrossRefGoogle Scholar
  13. Frigerio L, Pastres A, Prada A, Vitale (2001) Influence of KDEL on the fate of trimeric or assembly-defective phaseolin: selection use of an alternative route to vacuoles. Plant Cell 13:1109–1126CrossRefPubMedGoogle Scholar
  14. Gouy M, Gautier C (1982) Codon usage in bacteria: correlation with gene expressivity. Nucl Acids Res 10:7055–7074PubMedCrossRefGoogle Scholar
  15. Graf E (1983) Calcium binding to phytic acid. J Agri Food Chem 31:851–855CrossRefGoogle Scholar
  16. Graf E, Eaton JW (1990) Antioxidant functions of phytic acid. Free Radic Biol 8(1):61–69CrossRefGoogle Scholar
  17. Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression.Trends Biotechnol 22:346–353CrossRefPubMedGoogle Scholar
  18. Hadlington JL, Denecke J (2000) Sorting of soluble proteins in the secretory pathway of plants. Curr Opin Plant Biol 3:461–468CrossRefPubMedGoogle Scholar
  19. Herman EM, Tague BW, Hoffman LM, Kjemtrup SE, Chrispeels MJ (1990) Retention of phytohemagglutinin with carboxyterminal tetrapeptide KDEL in the nuclear envelope and the endoplasmic reticulum. Planta 182:305–312CrossRefGoogle Scholar
  20. Hong CY, Cheng KJ, Tseng TH, Wang CS, Liu LF, Yu SM (2004) Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds. Transgenic Res 13:29–39CrossRefPubMedGoogle Scholar
  21. Ikemura T (1981) Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol 151:389–409CrossRefPubMedGoogle Scholar
  22. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405Google Scholar
  23. Kusnadi AR, Hood EE, Witcher DR, Howard JA, Nikolov ZL (1998) Production and purification of two recombinant proteins from transgenic corn. Biotechnol Prog 14:149–55CrossRefPubMedGoogle Scholar
  24. Lagercrantz U (1998) Comparative mapping between Arabidopsis thaliana and Brassica nigra indicates that Brassica genomes have evolved through extensive genome replication accompanied by chromosome fusions and frequent rearrangements. Genetics 150:1217–1228PubMedGoogle Scholar
  25. Lei XG, Stahl CH (2001) Biotechnological development of effective phytases for mineral nutrition and environmental protection. Appl Microbiol Biotechnol 57:474–481CrossRefPubMedGoogle Scholar
  26. Li J, Hegeman CE, Hanlon RW, Lacy GH, Denbow MD, Grabau EA (1997) Secretion of active recombinant phytase from soybean cell-suspension cultures. Plant Physiol 114:1103–1111CrossRefPubMedGoogle Scholar
  27. Ma JK, Hein MB (1995) Immunotherapeutic potential of antibodies produced in plants. Trends Biotechnol 13:522–527CrossRefPubMedGoogle Scholar
  28. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiol 15:473–479CrossRefGoogle Scholar
  29. Nambiar KP, Stackhouse J, Stauffer DM, Kennedy WP, Eldredge JK, Benner SA (1984) Total synthesis and cloning of a gene coding for the ribonuclease S protein. Science 223:1299–1301PubMedCrossRefGoogle Scholar
  30. Napier JA, Richard G, Turner MFP, Shewry PR (1997) Trafficking of wheat gluten proteins in transgenic tobacco plants: γ-Gliadin does not contain an endoplasmic reticulum-retention signal. Planta 203:488–494CrossRefPubMedGoogle Scholar
  31. Pen J, Verwoerd TC, van Paridon PA, Beudeker RF, van den Elzen PJM, Geerse K, van der Klis JD, Versteegh HAJ, van Ooyen AJJ, Hoekema A (1993) Phytase-containing transgenic sees as a novel feed additive for improved phosphorus utilization. Biotechnology 11:811–814CrossRefGoogle Scholar
  32. Peng RH, Huang XM, Li X, Shun AJ, Yao QH, Peng YL (2001) Construction of plant binary expression vector containing intron-kanamycin gene and transformation in Nictiana tabacum. Acta Phytophysiol Sin 27:55–60Google Scholar
  33. Peng RH, Xiong AS, Li X, Fuan HQ, Yao QH (2003) A delta-endotoxin encoded in Pseudomonas fluorescens displays a high degree of insecticidal activity. Appl Microbiol Biotechnol 63:300–306CrossRefPubMedGoogle Scholar
  34. Plesse B, Durr A, Marbach J, Genschik P, Fleck J (1997) Identification of a new cis-regulatory element in a Nicotiana tabacum polyubiquitin gene promoter. Mol Gen Genet 254:258–266CrossRefPubMedGoogle Scholar
  35. Ponstein AS, Bade JB, Verwoerd TC, Molendijk L, Storms J, Beudeker RF, Pen J (2002) Stable expression of Phytase (phyA) in canola (Brassica napus) seeds: towards a commercial product. Mol Breed 10:31–44CrossRefGoogle Scholar
  36. Raboy V (1997) Low phytic acid mutants and selection thereof. US patent 5,689,504 US PTOGoogle Scholar
  37. Reddy NR, Sathe SK, Salunkhe DK (1982) Phytases in legumes and cereals. Adv Food Res 28:1–92PubMedGoogle Scholar
  38. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Harbor, NYGoogle Scholar
  39. Simons PC, Versteegh HA, Jongbloed AW, Kemme PA, Slump P, Bos KD, Wolters MG, Beudeker RF, Verschoor GJ (1990) Improvement of phosphorus availability by microbial phytase in broilers and pigs. Br J Nutr 64:525–540CrossRefPubMedGoogle Scholar
  40. Stoger E, Vaquero C, Torres E, Sack M, Nicholson U, Drossard J, Williams S, Keen D, Perrin Y, Verwoerd TC, Dekker BM, Hoekema A (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17:2362PubMedCrossRefGoogle Scholar
  41. Sugimoto K, Otsuki Y, Saji S, Hirochika H (1994) Transposition of the maize Ds element from a viral vector to the rice genome. Plant J 5:863–871CrossRefPubMedGoogle Scholar
  42. Sun CC, Zhao H, Fang GH, Wang WR, Li YL, Qian XF (2000) Study on pure-breeding techniques of some rapeseed cultivars (Brassica napus L) with low erucic acid and low glucosinolates in Shanghai's ecological condition. Acta Agric Shanghai 16:45–49Google Scholar
  43. Tanaka A, Mita S, Ohta S, Kyozuka J, Shimamoto K, Nakamura K (1990) Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron. Nucleic Acids Res 18:6767–6770PubMedCrossRefGoogle Scholar
  44. Ullah AH, Sethumadhavan K, Mullaney EJ, Ziegelhoffer T, Austin-Phillips S (2002) Cloned and expressed fungal phyA gene in alfalfa produces a stable phytase. Biochem Biophys Res Commun 290:1343–1348CrossRefPubMedGoogle Scholar
  45. van Hartingsveldt W, van Zeijl CM, Hareveld GM, Gouka RJ Suykerbuyk ME, Luiten RG, van Paridon PA, Selten GC, Veenstra AE (1993) Cloning, characterization and over-expression of the phytase-encoding gene (phyA) of Aspergillus niger. Gene 127:87–94CrossRefPubMedGoogle Scholar
  46. Verwoerd TC, van Paridon PA PA, van Ooyen AJ, van Lent JW, Hoekema A, Pen J (1995) Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves. Plant Physiol 109:1199–1205CrossRefPubMedGoogle Scholar
  47. Wandelt CI, Khan MR, Craig S, Schroeder HE, Spencer D, Higgins TJ (1992) Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant J 2:181–192PubMedGoogle Scholar
  48. Whitelam GC, Cockburn W, Owen MR (1994) Antibody production in transgenic plants. Biochem Soc Trans 22:940–944.PubMedGoogle Scholar
  49. Xiong AS, Yao HQ, Peng RH, Li X, Fan QH, Li Y, Cheng ZM (2004) A simple, rapid, high fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for long gene sequences. Nucl Acids Res 32:e98CrossRefPubMedGoogle Scholar
  50. Yao QH, Huang XM, Peng RH (1999) Synthesis and sequence determination of ACC deaminase gene using successive expressive extension PCR method. Acta Agric Shanghai (Suppl) 15:11–16Google Scholar
  51. Zhang ZB, Kornegay ET, Radcliffe JS, Denbow DM, Veit HP, Larsen CT (2000a) Comparison of genetically engineered microbial and plant phytases for young broilers. Poultry Sci 79:709–717Google Scholar
  52. Zhang ZB, Kornegay ET, Radcliffe JS, Wilson JH, Veit HP (2000b) Comparison of genetically engineered Aspergillus and canola in weaning pig diets. J Anim Sci 78:2868–2878PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Ri-He Peng
    • 1
  • Quan-Hong Yao
    • 1
  • Ai-Sheng Xiong
    • 1
  • Zong-Ming Cheng
    • 2
  • Yi Li
    • 3
  1. 1.Shanghai Key Laboratory of Agricultural Genetics and BreedingAgro-Biotechnology Research Center, Shanghai Academy of Agricultural SciencesShanghaiPeople's Republic China
  2. 2.Department of Plant SciencesUniversity of TennesseeKnoxvilleUSA
  3. 3.Department of Plant ScienceUniversity of ConnecticutStorrsUSA

Personalised recommendations