Plant Cell Reports

, Volume 32, Issue 4, pp 529–536 | Cite as

An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin

  • Natsuko Kurokawa
  • Tadayoshi Hirai
  • Mariko Takayama
  • Kyoko Hiwasa-Tanase
  • Hiroshi Ezura
Original Paper


Key message

The E8 promoter–HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit.


Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8–MIR–HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30–630 μg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8–MIR–HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113–124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.


Expression vector E8 promoter HSP terminator Miraculin Transgenic tomato plants 



We thank the members of the Ezura laboratory for their helpful discussions. This study was supported by the project ‘‘Development of Fundamental Technologies for the Production of High-value Materials Using Transgenic Plants’’ by the Ministry of Economy, Trade, and Industry of Japan (to H.E.). The Micro-Tom seeds (TOMJPF00001) were provided by the National BioResource Project (NBRP) of the MEXT, Japan.


  1. Bhattacharyya J, Chowdhury AH, Ray S, Jha JK, Das S, Gayen S, Chakraborty A, Mitra J, Maiti MK, Basu A, Sen SK (2012) Native polyubiquitin promoter of rice provides increased constitutive expression in stable transgenic rice plants. Plant Cell Rep 31:271–279. doi: 10.1007/s00299-011-1161-4 PubMedCrossRefGoogle Scholar
  2. Cheon BY, Kim HJ, Oh KH, Bahn SC, Ahn JH, Choi JW, Ok SH, Bae JM, Shin JS (2004) Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis. Transgenic Res 13:541–549. doi: 10.1007/s11248-004-2737-3 PubMedCrossRefGoogle Scholar
  3. Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes- structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689. doi: 10.1007/bf00020010 PubMedCrossRefGoogle Scholar
  4. Dai ZY, Hooker BS, Anderson DB, Thomas SR (2000) Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects. Transgenic Res 9:43–54. doi: 10.1023/a:1008922404834 PubMedCrossRefGoogle Scholar
  5. Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends in Plant Sci 6:219–226. doi: 10.1016/s1360-1385(01)01922-7 CrossRefGoogle Scholar
  6. Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, van Montagu M, Leemans J (1985) Efficent octopine Ti plasmid derived vectors for Agrobacterium-mediated gene transfer in plants. Nucleic Acids Res 13:4777–4785PubMedCrossRefGoogle Scholar
  7. 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 PubMedCrossRefGoogle Scholar
  8. 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 PubMedCrossRefGoogle Scholar
  9. He ZM, Jiang XL, Qi Y, Luo DQ (2008) Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression. Genetica 133:207–214. doi: 10.1007/s10709-007-9201-2 PubMedCrossRefGoogle Scholar
  10. Hirai T, Kim YW, Kato K, Hiwasa-Tanase K, Ezura H (2011a) Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter. Transgenic Res 20:1285–1292. doi: 10.1007/s11248-011-9495-9 PubMedCrossRefGoogle Scholar
  11. Hirai T, Kurokawa N, Duhita N, Hiwasa-Tanase K, Kato K, Ezura H (2011b) The HSP terminator of Arabidopsis thaliana induces a high level of miraculin accumulation in transgenic tomatoes. J Agric Food Chem 59:9942–9949. doi: 10.1021/jf202501e PubMedCrossRefGoogle Scholar
  12. Hiwasa-Tanase K, Nyarubona M, Hirai T, Kato K, Ichikawa T, Ezura H (2011) 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. Plant Cell Rep 30:113–124. doi: 10.1007/s00299-010-0949-y PubMedCrossRefGoogle Scholar
  13. Jiang XL, He ZM, Peng ZQ, Qi Y, Chen Q, Yu SY (2007) Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice. Transgenic Res 16:169–175. doi: 10.1007/s11248-006-9023-5 PubMedCrossRefGoogle Scholar
  14. Kato K, Yoshida R, Kikuzaki A, Hirai T, Kuroda H, Hiwasa-Tanase K, Takane K, Ezura H, Mizoguchi T (2010) Molecular breeding of tomato lines for mass production of miraculin in a plant factory. J Agric Food Chem 58:9505–9510. doi: 10.1021/jf101874b PubMedCrossRefGoogle Scholar
  15. Kim YW, Kato K, Hirai T, Hiwasa-Tanase K, Ezura H (2010a) Spatial and developmental profiling of miraculin accumulation in transgenic tomato fruits expressing the miraculin gene constitutively. J Agric Food Chem 58:282–286. doi: 10.1021/jf9030663 PubMedCrossRefGoogle Scholar
  16. Kim YW, Hirai T, Kato K, Hiwasa-Tanase K, Ezura H (2010b) Gene dosage and genetic background affect miraculin accumulation in transgenic tomato fruits. Plant Biotechnol 27:333–338. doi: 10.5511/plantbiotechnology.27.333 CrossRefGoogle Scholar
  17. Kurihara K, Beidler LM (1968) Taste-modifying protein from miracle fruit. Science 161:1241–1243PubMedCrossRefGoogle Scholar
  18. Leclercq J, Ranty B, Sanchez-Ballesta MT, Li ZG, Jones B, Jauneau A, Pech JC, Latche A, Ranjeva R, Bouzayen M (2005) Molecular and biochemical characterization of LeCRK1, a ripening-associated tomato CDPK-related kinase. J Exp Bot 56:25–35. doi: 10.1093/jxb/eri003 PubMedGoogle Scholar
  19. Matsuoka M, Tada Y, Fujimura T, Kanomurakami Y (1993) Tissue-specific light-regulated expression directed by the promoter of a C4 gene, mayze pyruvate, orthophosphate dikinase, in a C3 plant, rice. Proc Natl Acad Sci USA 90:9586–9590. doi: 10.1073/pnas.90.20.9586 PubMedCrossRefGoogle Scholar
  20. 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 suspension cells. J Biosci Bioeng 105:39–47. doi: 10.1263/jbb.105.39 PubMedCrossRefGoogle Scholar
  21. Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y, Ohashi Y (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37:49–59PubMedCrossRefGoogle Scholar
  22. 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 PubMedCrossRefGoogle Scholar
  23. Outchkourov NS, Peters J, de Jong J, Rademakers W, Jongsma MA (2003) The promoter-terminator of chrysanthemum rbcS1 directs very high expression levels in plants. Planta 216:1003–1012. doi: 10.1007/s00425-002-0953-8 PubMedGoogle Scholar
  24. Rasmussen TB, Donaldson IA (2006) Investigation of the endosperm-specific sucrose synthase promoter from rice using transient expression of reporter genes in guar seed tissue. Plant Cell Rep 25:1035–1042. doi: 10.1007/s00299-006-0158-x PubMedCrossRefGoogle Scholar
  25. 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. doi: 10.1023/a:1008979525909 PubMedCrossRefGoogle Scholar
  26. Satoh J, Kato K, Shinmyo A (2004) The 5 ‘-untranslated region of the tobacco Alcohol dehydrogenase gene functions as an effective translational enhancer in plant. J Biosci Bioeng 98:1–8. doi: 10.1016/s1389-1723(04)70234-0 PubMedGoogle Scholar
  27. Streatfield SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 5:2–15. doi: 10.1111/j.1467-7652.2006.00216.x PubMedCrossRefGoogle Scholar
  28. Sugaya T, Yano M, Sun HJ, Hirai T, Ezura H (2008) Transgenic strawberry expressing the taste-modifying protein miraculin. Plant Biotechnol 25:329–333. doi: 10.5511/plantbiotechnology.25.329 CrossRefGoogle Scholar
  29. Sugio T, Satoh J, Matsuura H, Shinmyo A, Kato K (2008) The 5 ‘-untranslated region of the Oryza sativa alcohol dehydrogenase gene functions as a translational enhancer in monocotyledonous plant cells. J Biosci Bioeng 105:300–302. doi: 10.1263/jbb.105.300 PubMedCrossRefGoogle Scholar
  30. 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 PubMedCrossRefGoogle Scholar
  31. 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 PubMedCrossRefGoogle Scholar
  32. 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 PubMedCrossRefGoogle Scholar
  33. 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
  34. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R (2003) Molecular farming in plants: host systems and expression technology. Trends in Biotechnol 21:570–578. doi: 10.1016/j.tibtech.2003.10.002 CrossRefGoogle Scholar
  35. Yano M, Hirai T, Kato K, Hiwasa-Tanase K, Fukuda N, Ezura H (2010) Tomato is a suitable material for producing recombinant miraculin genetically stable manner. Plant Sci 178:469–473. doi: 10.1016/j.plantsci.2010.02.016 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Natsuko Kurokawa
    • 1
  • Tadayoshi Hirai
    • 1
  • Mariko Takayama
    • 1
  • Kyoko Hiwasa-Tanase
    • 1
  • Hiroshi Ezura
    • 1
  1. 1.Graduate School of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan

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