Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

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.

Abstract

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.

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

Access this article

Log in via an institution

We’re sorry, something doesn't seem to be working properly.

Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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 13:4777–4785

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Kurihara K, Beidler LM (1968) Taste-modifying protein from miracle fruit. Science 161:1241–1243

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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 37:49–59

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  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. doi:10.1023/a:1008979525909

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Theerasilp S, Kurihara Y (1988) Complete purification and characterization of the taste-modifying protein, miraculin, from miracle fruit. J Biol Chem 263:11536–11539

    PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki, 305-8572, Japan

    Natsuko Kurokawa, Tadayoshi Hirai, Mariko Takayama, Kyoko Hiwasa-Tanase & Hiroshi Ezura

Authors
  1. Natsuko Kurokawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tadayoshi Hirai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mariko Takayama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kyoko Hiwasa-Tanase
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hiroshi Ezura
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hiroshi Ezura.

Additional information

Communicated by H. Ebinuma.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kurokawa, N., Hirai, T., Takayama, M. et al. An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin. Plant Cell Rep 32, 529–536 (2013). https://doi.org/10.1007/s00299-013-1384-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-013-1384-7

Keywords

Search

Navigation