Plant Cell Reports

, Volume 30, Issue 1, pp 113–124 | Cite as

High-level accumulation of recombinant miraculin protein in transgenic tomatoes expressing a synthetic miraculin gene with optimized codon usage terminated by the native miraculin terminator

  • Kyoko Hiwasa-Tanase
  • Mpanja Nyarubona
  • Tadayoshi Hirai
  • Kazuhisa Kato
  • Takanari Ichikawa
  • Hiroshi Ezura
Original Paper

Abstract

In our previous study, a transgenic tomato line that expressed the MIR gene under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator (tNOS) produced the taste-modifying protein miraculin (MIR). However, the concentration of MIR in the tomatoes was lower than that in the MIR gene’s native miracle fruit. To increase MIR production, the native MIR terminator (tMIR) was used and a synthetic gene encoding MIR protein (sMIR) was designed to optimize its codon usage for tomato. Four different combinations of these genes and terminators (MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR) were constructed and used for transformation. The average MIR concentrations in MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR fruits were 131, 197, 128 and 287 μg/g fresh weight, respectively. The MIR concentrations using tMIR were higher than those using tNOS. The highest MIR accumulation was detected in sMIR-tMIR fruits. On the other hand, the MIR concentration was largely unaffected by sMIR-tNOS. The expression levels of both MIR and sMIR mRNAs terminated by tMIR tended to be higher than those terminated by tNOS. Read-through mRNA transcripts terminated by tNOS were much longer than those terminated by tMIR. These results suggest that tMIR enhances mRNA expression and permits the multiplier effect of optimized codon usage.

Keywords

Miraculin Codon optimization Miraculin terminator Transgenic tomato Read-through 

Abbreviations

GUS

β-Glucronidase

MIR

Miraculin

sMIR

Synthesized MIR

NOS

Nopaline synthase

References

  1. Batard Y, Hehn A, Nedelkina S, Schalk M, Pallett K, Schaller H, Werck-Reichhart D (2000) Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems. Arch Biochem Biophys 379:161–169. doi:10.1006/abbi.2000.1867 CrossRefPubMedGoogle Scholar
  2. Chiba Y, Ishikawa M, Kijima F, Tyson RH, Kim J, Yamamoto A, Nambara E, Leustek T, Wallsgrove RM, Naito S (1999) Evidence for autoregulation of cystathionine γ-synthase mRNA stability in Arabidopsis. Science 286:1371–1374. doi:10.1126/science.286.5443.1371 CrossRefPubMedGoogle Scholar
  3. Chincinska IA, Liesche J, Krugel U, Michalska J, Geigenberger P, Grimm B, Khun C (2008) Sucrose transporter StSUT4 from potato affects flowering, tuberization, and shade avoidance response. Plant Physiol 146:515–528. doi:10.1104/pp.107.112334 CrossRefPubMedGoogle Scholar
  4. Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6:219–226. doi:10.1016/S1360-1385(01)01922-7 CrossRefPubMedGoogle Scholar
  5. Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, van Montagu M, Leemans J (1985) Efficient octopine Ti plasmid derived vectors for Agrobacterium-mediated gene transfer in plants. Nucleic Acids Res 13:4777–4785CrossRefPubMedGoogle Scholar
  6. Desai PN, Shrivastava N, Padh H (2010) Production of heterologous proteins in plants: Strategies for optimal expression. Biotechnol Adv 28:427–435. doi:10.1016/j.biotechadv.2010.01.005 CrossRefPubMedGoogle Scholar
  7. Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18:1151–1155. doi:10.1038/81132 CrossRefPubMedGoogle Scholar
  8. Gilmartin GM (2005) Eukaryotic mRNA 3′ processing: a common means to different ends. Genes Dev 19:2517–2521. doi:10.1101/gad.1378105 CrossRefPubMedGoogle Scholar
  9. Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22:346–353. doi:10.1016/j.tibtech.2004.04.006 CrossRefPubMedGoogle Scholar
  10. Gutiérrez RA, Maclntosh GC, Green PJ (1999) Current perspectives on mRNA stability in plants: multiple levels and mechanisms of control. Trend Plant Sci 4:429–438. doi:10.1016/S1360-1385(99)01484-3 CrossRefGoogle Scholar
  11. Hackel A, Schauer N, Carrari F, Fernie AR, Grimm B, Kuhn C (2006) Sucrose transporter LeSUT1 and LeSUT2 inhibition affects tomato fruit development in different ways. Plant J 45:180–192. doi:10.1111/j.1365-313X.2005.02572.x CrossRefPubMedGoogle Scholar
  12. Hirai T, Fukukawa G, Kakuta H, Fukuda N, Ezura H (2010) Production of recombinant miraculin using transgenic tomatoes in a closed cultivation system. J Agric Food Chem 58:6096–6101. doi:10.1021/jf100414v CrossRefPubMedGoogle Scholar
  13. Ingelbrecht ILW, Herman LMF, Dekeyser RA, Van Montagu MC, Depicker AG (1989) Different 3′ end regions strongly influence the level of gene expression in plant cells. Plant Cell 1:671–680. doi:10.1105/tpc.1.7.671 CrossRefPubMedGoogle Scholar
  14. Ingelbrecht ILW, Breyne P, Vancompernolle K, Jacobs A, Van Montagu MC, Depicker AG (1991) Transcriptional interference in transgenic plants. Gene 109:239–243. doi:10.1016/0378-1119(91)90614-H CrossRefPubMedGoogle Scholar
  15. Jackson RJ, Standart N (1990) Do the poly (A) tail and 3′ untranslated region control mRNA translation? Cell 62:15–24. doi:10.1016/0092-8674(90)90235-7 CrossRefPubMedGoogle Scholar
  16. Jefferson RK, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  17. Kawabe A, Miyashita NT (2003) Patterns of codon usage bias in three dicot and four monocot plant species. Genes Genet Syst 78:343–352. doi:10.1266/ggs.78.343 CrossRefPubMedGoogle Scholar
  18. Kim YW, Kato K, Hirai T, Hiwasa-Tanase K, Ezura H (2010) Spatial and development profiling of miraculin accumulation in transgenic tomato fruits expressing the miraculin gene constitutively. J Agric Food Chem 58:282–286. doi:10.1021/jf9030663 CrossRefPubMedGoogle Scholar
  19. Liu Q, Xue Q (2005) Comparative studies on codon usage pattern of chloroplasts and their host nuclear genes in four plant species. J Genet 84:55–62CrossRefPubMedGoogle Scholar
  20. Mason HS, Warzecha H, Mor T, Arntzen CJ (2002) Edible plant vaccines: application for prophylactic and molecular medicine. Trends Mol Med 8:324–329. doi:10.1016/S1471-4914(02)02360-2 CrossRefPubMedGoogle Scholar
  21. Matsuura H, Shinmyo A, Kato K (2008) Preferential translation mediated by Hsp81–3 5′-UTR during heat shock involves ribosome entry at the 5′-end rather than an internal site in Arabidopsis suspention cells. J Biosci Bioeng 105:39–47CrossRefPubMedGoogle Scholar
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
  23. Murray EE, Lotzer J, Eberle M (1989) Codon usage in plant genes. Nucleic Acids Res 17:477–498CrossRefPubMedGoogle Scholar
  24. Nagaya S, Kawamura K, Shinmyo A, Kato K (2010) The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells. Plant Cell Physiol 51:328–332. doi:10.1093/pcp/pcp188 CrossRefPubMedGoogle Scholar
  25. Peng RH, Yao QH, Xiong AS, Cheng ZM, Li Y (2006) Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola. Plant Cell Rep 25:124–132. doi:10.1007/s00299-005-0036-y CrossRefPubMedGoogle Scholar
  26. Perlak FJ, Fuchs RL, Dean DA, McPherson SL, Fischhoff DA (1991) Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci USA 88:3324–3328CrossRefPubMedGoogle Scholar
  27. Proudfoot N (2004) New perspectives on connecting messenger RNA 3′ end formation to transcription. Curr Opin Cell Biol 16:272–278. doi:10.1016/j.ceb.2004.03.007 CrossRefPubMedGoogle Scholar
  28. Rang A, Linke B, Jansen B (2005) Detection of RNA variants transcribed from the transgene in Roundup Ready soybean. Eur Food Res Technol 220:438–443. doi:10.1007/s00217-004-1064-5 CrossRefGoogle Scholar
  29. Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5:69–76. doi:10.1007/BF00020088 CrossRefGoogle Scholar
  30. Rothnie HM, Reid J, Hohn T (1994) The contribution of AAUAAA and the upstream element UUUGUA to the efficiency of mRNA 3′-end formation in plants. EMBO J 13:2200–2210PubMedGoogle Scholar
  31. Rouwendal GJ, Mendes O, Wolbert EJ, de Boer AD (1997) Enhanced expression in tobacco of the gene encoding green fluorescent protein by modification of its codon usage. Plant Mol Biol 33:989–999. doi:10.1023/A:1005740823703 CrossRefPubMedGoogle Scholar
  32. Shen WJ, Forde BG (1989) Efficient transformation of Agrobacterium spp. by high voltage electroporation. Nucleic Acids Res 17:8385CrossRefPubMedGoogle Scholar
  33. Sugaya T, Yano M, Sun HJ, Hirai T, Ezura H (2008) Transgenic strawberry expressing a taste-modifying protein, miraculin. Plant Biotechnol 25:329–333Google Scholar
  34. Sun HJ, Cui ML, Ma B, Ezura H (2006a) Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce. FEBS Lett 580:620–626. doi:10.1016/j.febslet.2005.12.080 CrossRefPubMedGoogle Scholar
  35. Sun HJ, Uchii S, Watanabe S, Ezura H (2006b) A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol 47:426–431. doi:10.1093/pcp/pci251 CrossRefPubMedGoogle Scholar
  36. Sun HJ, Kataoka H, Yano M, Ezura H (2007) Genetically stable expression of functional miraculin, a new type of alternative sweetener, in transgenic tomato plants. Plant Biotechnol J 5:768–777. doi:10.1111/j.1467-7652.2007.00283.x CrossRefPubMedGoogle Scholar
  37. Suzuki A, Shirata Y, Ishida H, Chiba Y, Onouchi H, Naito S (2001) The first exon coding region of cystathionine γ-synthase gene is necessary and sufficient for downregulation of its own mRNA Accumulation in transgenic Arabidopsis thaliana. Plant Cell Physiol 42:1174–1180CrossRefPubMedGoogle Scholar
  38. Theerasilp S, Kurihara Y (1988) Complete purification and characterization of the taste-modifying protein, miraculin, from miracle fruit. J Biol Chem 263:11536–11539PubMedGoogle Scholar
  39. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R (2003) Molecular farming in plants: host systems and expression technology. Trends Biotechnol 21:570–578. doi:10.1016/j.tibtech.2003.10.002 CrossRefPubMedGoogle Scholar
  40. Windels P, Taverniers I, Depicker A, Van Bockstaele E, De Loose M (2001) Characterisation of the Roundup Ready soybean insert. Eur Food Res Technol 213:107–112CrossRefGoogle Scholar
  41. Xue GP, Patel M, Johnson JS, Smyth DJ, Vickers CE (2003) Selectable marker-free transgenic barley producing a high level of cellulose (1, 4-β-glucanase) in developing grains. Plant Cell Rep 21:1088–1094. doi:10.1007/s00299-003-0627-4 CrossRefPubMedGoogle Scholar
  42. Yano M, Hirai T, Kato K, Hiwasa-Tanase K, Fukuda N, Ezura H (2010) Tomato is a suitable material for producing recombinant miraculin protein in genetically stable manner. Plant Sci 178:469–473. doi:10.1016/j.plantsci.2010.02.016 CrossRefGoogle Scholar
  43. Zarudnaya MI, Kolomiets M, Potyahaylo AL, Hovorun DM (2003) Downstream elements of mammalian pre-mRNA polyadenylation signals: primary, secondary and higher-order structures. Nucleic Acids Res 31:1375–1386CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Kyoko Hiwasa-Tanase
    • 1
  • Mpanja Nyarubona
    • 1
  • Tadayoshi Hirai
    • 1
  • Kazuhisa Kato
    • 1
  • Takanari Ichikawa
    • 2
  • Hiroshi Ezura
    • 1
  1. 1.Graduate School of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan
  2. 2.Scientific Senior Manager of Technology Center, Team Leader of Accreditation and Evaluation Team for the New UniversityOkinawa Institute of Science and Technology Promotion CorporationOnna-sonJapan

Personalised recommendations