Transgenic Research

, Volume 14, Issue 5, pp 677–684 | Cite as

Expression of the Small Peptide GLP-1 in Transgenic Plants

  • Hiroshi Yasuda
  • Yoshifumi Tada
  • Yuji Hayashi
  • Takahito Jomori
  • Fumio Takaiwa
Article

Abstract

Glucagon-like peptide 1 (GLP-1) has great potential in diabetes therapy. In order to accumulate GLP-1 in endosperm tissue of rice, a codon-optimized GLP-1 (mGLP-1) synthetic gene was directly expressed under the control of rice storage protein glutelin GluB-1 promoter in transgenic rice plants. Unexpectedly, neither the transcripts nor the transgene products were detected in the seeds of regenerated plants. Furthermore, transcripts of GluB-1 gene in these transgenic plants were not detected, and small interfering RNAs (siRNAs) corresponding to the transgene were detected. These results indicated that the expression of mGLP-1 was silenced by co-suppression in rice transgenic seeds. To avoid silencing, mGLP-1 was fused to GFP with or without self-processing 2A sequence, and introduced into rice plants. Both chimeric genes were highly expressed in these transgenic rice seeds, indicating that gene silencing could be avoided by changing the transgene components. Furthermore, the fusion protein containing the 2A sequence were processed into GFP-2A and mGLP-1 peptides with the efficiency of more than 80%, but the processed mGLP-1 peptides were not detected. Lack of accumulation of mGLP-1 may be explained by proteolytic digestion in the cytoplasm.

Keywords

co-suppression GLP-1 2A sequence transgenic rice 

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References

  1. Becker, D, Kemper, E, Schell, J, Masterson, R 1992New plant binary vectors with selectable markers located proximal to the left T-DNA borderPlant Mol Biol2011951197CrossRefPubMedGoogle Scholar
  2. Bell, GI, Sanchez-Pescadore, R, Laybourn, PL, Najarian, RC 1983Exon duplication and divergence in the human preproglucagon geneNature304368371CrossRefPubMedGoogle Scholar
  3. Donnelly, MLL, Luke, G, Mehrotra, A, Li, X, Hughes, LE, Gani, D, Ryan, MD 2001Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’J Gen Virol8210131025PubMedGoogle Scholar
  4. Drucker, DJ, Philippe, J, Mojsov, S, Chick, WL, Habener, JF 1987Glucagon-like peptide 1 stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell lineProc Natl Acad Sci USA8434343438PubMedGoogle Scholar
  5. Erickson, L, Yu, W-J, Zhang, J, Lange, CFM, McBride, B, Du, S 2002

    The Production and Delivery of Therapeutic Peptides in Plants

    Erickson, LYu, W-JBrandle, JRymerson, R eds. Molecular Farming of Plants and Animals for Human and Veterinary MedicineKluwer Academic PublishersDordrecht, Netherlands197222
    Google Scholar
  6. Francois, IEJA, Bolle, MFC, Dwyer, G, Goderis, IJWM, Woutors, PFJ, Verhaert, PD, Proost, P, Schaaper, WMM, Cammue, BPA, Broekaert, WF 2002Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteinsPlant Physiol12813461358CrossRefPubMedGoogle Scholar
  7. Goto, F, Yoshihara, T, Shigemoto, N, Toki, S, Takaiwa, F 1999Iron fortification of rice seed by the soybean ferritin geneNat Biotechnol17282286CrossRefPubMedGoogle Scholar
  8. Halpin, C, Cooke, SE, Barakate, A, Amrani, AE, Ryan, MD 1999Self-processing 2A-polyproteins–a system for co-ordinate expression of multiple proteins in transgenic plantsPlant J17453459CrossRefPubMedGoogle Scholar
  9. Hamilton, A, Voinnet, O, Chappell, L, Baulcombe, D 2002Two classes of short interfering RNA in RNA silencingEMBO J2146714679CrossRefPubMedGoogle Scholar
  10. Holst, JJ 1983Gut glucagon, enteroglucagon, gut glucagonlike immunoreactivity, glicentin–current statusGastroenterology8416021613PubMedGoogle Scholar
  11. Iida, S, Amano, E, Nishio, T 1993A rice (Oryza sativa L.) mutant having a low content of glutelin and a high content of prolamineTheor Appl Genet87374378CrossRefGoogle Scholar
  12. Kusaba, M, Miyahara, K, Iida, S, Fukuoka, H, Takano, T, Sassa, H, Nishimura, M, Nishio, T 2003Low glutelin content1: A dominant mutation that suppresses the Glutelin multigene family via RNA silencing in ricePlant Cell1514551467CrossRefPubMedGoogle Scholar
  13. Larrick, JW, Yu, L, Naftzger, C, Jaiswal, S, Wycoff, K 2002

    Human Pharmaceuticals Produced in Plants

    Hood, EEHoward, JA eds. Plants as Factories for protein productionKluwer Academic PublishersDordrecht, Netherlands79101
    Google Scholar
  14. Ma, C, Mitra, A 2002Expressing multiple genes in a single open reading frame with the 2A region of foot-and-mouth disease virus as a linkerMol Breeding9191199CrossRefGoogle Scholar
  15. Mette, MF, Aufsatz, W, Winden, J, Matzke, MA, Matzke, AJM 2000Transcriptional silencing and promoter methylation triggered by double-stranded RNAEMBO J1951945201CrossRefPubMedGoogle Scholar
  16. Murata, Y, Ueki, J, Saito, H, Nitta, N, Imaseki, H 2001Transgenic rice with reduced glutelin content by transformation with glutelin A antisence geneMol Breeding8273284CrossRefGoogle Scholar
  17. Okamoto, M, Mitsuhara, I, Ohshima, M, Natori, S, Ohashi, Y 1998Enhanced expression of an antimicrobial peptide Sarcotoxin IA by GUS fusion in transgenic tobacco plantsPlant Cell Physiol395763PubMedGoogle Scholar
  18. Qu, LQ, Takaiwa, F 2004Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic ricePlant Biotechnol J2113125CrossRefGoogle Scholar
  19. Rogers, SO, Bendich, AJ 1988

    Extraction of DNA from plant tissues

    Gelvin, SBSchilperoort, RA eds. Plant Molecular Biology Manual A6Kluwer Academic PublishersDordrecht, Netherlands110
    Google Scholar
  20. Ryan, MD, Drew, J 1994Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyproteinEMBO J13928933PubMedGoogle Scholar
  21. Schagger, H, Jagow, G 1987Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDaAnal Biochem166368379CrossRefPubMedGoogle Scholar
  22. Tada, Y, Utsumi, S, Takaiwa, F 2003Foreign gene products can be enhanced by introduction into low storage protein mutantsPlant Biotechnol J1411422CrossRefGoogle Scholar
  23. Takaiwa, F, Kikuchi, S, Oono, K 1987A rice glutelin gene family – A major type of glutelin mRNAs can be divided into two classesMol Gen Genet2081522CrossRefGoogle Scholar
  24. Takaiwa, F, Oono, K, Wing, D, Kato, A 1991Sequence of three members and expression of a new major subfamily of glutelin genes from ricePlant Mol Biol17875885CrossRefPubMedGoogle Scholar
  25. Weir, GC, Mojsov, S, Hendrick, GK, Hebener, JF 1989Glucagon-like peptide 1 (7–37) actions on endocrine pancreasDiabetes38338342PubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Hiroshi Yasuda
    • 1
  • Yoshifumi Tada
    • 1
  • Yuji Hayashi
    • 2
  • Takahito Jomori
    • 2
  • Fumio Takaiwa
    • 1
  1. 1.Department of Plant BiotechnologyNational Institute of Agrobiological SciencesIbarakiJapan
  2. 2.Sanwa Kagaku Kenkyusyo Co Ltd.Hokusei, MieJapan

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