Advertisement

Transgenic Research

, Volume 21, Issue 3, pp 545–553 | Cite as

Evaluation of seed storage protein gene 3′-untranslated regions in enhancing gene expression in transgenic rice seed

  • Wen Jing Li
  • Ling Ling Dai
  • Zhi Jian Chai
  • Zhi Jie Yin
  • Le Qing QuEmail author
Original Paper

Abstract

3′ untranslated regions (UTRs) are important sequence elements that modulate the expression of genes. We evaluated the potential of the 3′-UTRs of 9 seed storage protein (SSP) genes as terminators in enhancing the expression of the β-glucuronidase (gus A) reporter gene driven by the glutelin GluB-3 promoter in stable transgenic rice lines. Six of the 3′-UTRs significantly enhanced the activity of the GluB-3 promoter without changing its tissue specificity but altered its expression pattern in endosperm. With the 3′-UTRs of GluB-5, GluA-2 and GluC, the expression of gus A was higher by 3.12-, 2.45- and 2.14-fold, respectively, than with the Nos terminator. These three 3′-UTRs, combined with GluC, Ubi-1 and CaMV35S promoters, also increased GUS levels in stable transgenic rice lines or in transient expression in protoplasts, which indicated that the enhancements were independent of the promoter sequence. The increase in protein production was accompanied by altered mRNA levels, which suggests that the enhancements were due to increased transcript level. The 3′-UTRs of GluB-5, GluA-2 and GluC, when combined with strong promoters, might be ideal candidates for high production of recombinant proteins in rice seeds. The 9 SSP 3′-UTRs could function as faithful terminators in mono- or multi-gene transformation avoiding homology-based gene silencing.

Keywords

3′ untranslated region Expression level Seed storage protein Terminator Transgenic rice 

Notes

Acknowledgments

This work was supported by the Natural Science Foundation of China (No. 30871375) and the National Program of Transgenic Variety Development of China (2008ZX08009-004, 2008ZX08001-006).

Supplementary material

11248_2011_9552_MOESM1_ESM.pdf (16 kb)
Supplementary Fig. 1 GUS activities of T2 transgenic plants expressed by various 3′-UTRs downstream of the GluB-3 promoter in maturing seeds at 17 DAF. Horizontal bars represent the average GUS activity. pGluB-3: GluB-3 promoter; GluA-1, GluA-2, GluA-3, GluB-1, GluB-5, GluC, 16kD pro, Glb-1 and GluD-1 represent relative 3′-UTRs downstream of the GluB-3 promoter (PDF 15 kb)

References

  1. Ali S, Taylor W (2001a) The 3′ non-coding region of a C4 photosynthesis gene increases transgene expression when combined with heterologous promoters. Plant Mol Biol 46:325–333PubMedCrossRefGoogle Scholar
  2. Ali S, Taylor W (2001b) Quantitative regulation of the Flaveria Me1 gene is controlled by the 3′-untranslated region and sequences near the amino terminus. Plant Mol Biol 46:251–261PubMedCrossRefGoogle Scholar
  3. Anai T, Koga M, Tanaka H, Kinoshita T, Rahman SM, Takagi Y (2003) Improvement of rice (Oryza sativa L.) seed oil quality through introduction of a soybean microsomal ω-3 fatty acid desaturase gene. Plant Cell Rep 21:988–992PubMedCrossRefGoogle Scholar
  4. Atwater JA, Wisdom R, Verma IM (1990) Regulated mRNA stability. Annu Rev Genet 24:519–541PubMedCrossRefGoogle Scholar
  5. Bashirullah A, Cooperstock RL, Lipshitz HD (2001) Spatial and temporal control of RNA stability. Proc Natl Acad Sci USA 98:7025–7028PubMedCrossRefGoogle Scholar
  6. 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 18:675–689PubMedCrossRefGoogle Scholar
  7. De Jaeger G, Scheffer S, Jacobs A, Zambre M, Zobell O, Goossens A, Depicker A, Angenon G (2002) Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nat Biotechnol 20:1265–1268PubMedCrossRefGoogle Scholar
  8. Dean C, Favreau M, Bond-Nutter D, Bedbrook J, Dunsmuir P (1989) Sequences downstream of translation start regulate quantitative expression of two petunia RbcS genes. Plant Cell 1:201–208PubMedCrossRefGoogle Scholar
  9. Depicker A, Stachel S, Dhaese P, Zambryski P, Goodman HM (1982) Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet 1:561–573PubMedGoogle Scholar
  10. Guilley H, Dudley RK, Jonard G, Balazs E, Richards KE (1982) Transcription of cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of transcripts. Cell 30:763–770PubMedCrossRefGoogle Scholar
  11. Hobbs SLA, Warkentin TD, DeLong CMO (1993) Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol Biol 21:17–26PubMedCrossRefGoogle Scholar
  12. Hollams EM, Giles KM, Thomson AM, Leedman PJ (2002) mRNA stability and the control of gene expression: implications for human disease. Neurochem Res 27:957–980PubMedCrossRefGoogle Scholar
  13. Ingelbrecht ILW, Herman LMF, Dekeyser RA, Van Montagu MC, Depicker AG (1989) Different 3′ end regions strongly influence the leve1 of gene expression in plant cells. Plant Cell 1:671–680PubMedCrossRefGoogle Scholar
  14. Jing Q, Huang S, Guth S, Zarubin T, Motoyama A, Chen JM, Padova FD, Lin SC, Gram H, Han JH (2005) Involvement of MicroRNA in AU-Rich element-mediated mRNA instability. Cell 120:623–634PubMedCrossRefGoogle Scholar
  15. Kawakatsu T, Yamamoto MP, Hirose S, Yano M, Takaiwa F (2008) Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm. J Exp Bot 59:4233–4245PubMedCrossRefGoogle Scholar
  16. Knirsch L, Clerch LB (2000) A region in the 3′-UTR of MnSOD RNA enhances translation of a heterologous RNA. Biochem Biophys Res Commun 272:164–168PubMedCrossRefGoogle Scholar
  17. Lamacchia C, Shewry PR, Di Fonzo N, Forsyth JL, Harris N, Lazzeri PA, Napier JA, Halford NG, Barcelo P (2001) Endosperm specific activity of a storage protein gene promoter in transgenic wheat seed. J Exp Bot 52:243–250PubMedCrossRefGoogle Scholar
  18. Liu WX, Liu HL, Chai ZJ, Xu XP, Song YR, Qu LQ (2010) Evaluation of seed storage protein gene 5’-UTR in enhancing gene expression in transgenic rice seed. Thero Appl Genet 121:1267–1274CrossRefGoogle Scholar
  19. Merritt C, Rasoloson D, Ko D, Seydoux G (2008) 3′-UTRs are the primary regulators of gene expression in the C. elegans germline. Curr Biol 18:1476–1482PubMedCrossRefGoogle Scholar
  20. Monde RA, Greene JC, Stern DB (2000) The sequence and secondary structure of the 3′-UTR affect 3′-end maturation, RNA accumulation, and translation in tobacco chloroplasts. Plant Mol Biol 44:529–542PubMedCrossRefGoogle Scholar
  21. Ortega JL, Moguel-Esponda S, Potenza C, Conklin CF, Quintana A, Sengupta-Gopalan C (2006) The 3′ untranslated region of a soybean cytosolic glutamine synthetase (GS1) affects transcript stability and protein accumulation in transgenic alfalfa. Plant J 45:832–846PubMedCrossRefGoogle Scholar
  22. Qu LQ, Takaiwa F (2004) Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice. Plant Biotechnol J 2:113–125CrossRefGoogle Scholar
  23. Qu LQ, Yoshihara T, Ooyama A, Goto Y, Takaiwa F (2005) Iron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds. Planta 222:225–233CrossRefGoogle Scholar
  24. Qu LQ, Xing YP, Liu WX, Xu XP, Song YR (2008) Expression pattern and activity of six glutelin gene promoters in transgenic rice. J Exp Bot 59:2417–2424CrossRefGoogle Scholar
  25. Streatfield SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 5:2–15PubMedCrossRefGoogle Scholar
  26. Takagi H, Saito S, Yang L, Nagasaka S, Nishizawa N, Takaiwa F (2005) Oral immunotherapy against a pollen allergy using a seed-based peptide vaccine. Plant Biotechnol J 3:521–533PubMedCrossRefGoogle Scholar
  27. Takaiwa F, Takagi H, Hirose S, Wakasa Y (2007) Endosperm tissue is a good production platform for artificial recombinant proteins in transgenic rice. Plant Biotechnol J 5:84–92PubMedCrossRefGoogle Scholar
  28. Wakasa Y, Zhao H, Hirose S, Yamauchi D, Yamada Y, Yang L, Ohinata K, Yoshikawa M, Takaiwa F (2011) Antihypertensive activity of transgenic rice seed containing an 18-repeat novokinin peptide localized in the nucleolus of endosperm cells. Plant Biotechnol J doi: 10.1111/j.1467-7652.2010.00576.x
  29. Yang LJ, Tada Y, Yamamoto MP, Zhao H, Yoshikawa M, Takaiwa F (2006) A transgenic rice seed accumulating an antihypertensive peptide reduces the blood pressure of spontaneously hypertensive rats. FEBS Lett 580:3315–3320PubMedCrossRefGoogle Scholar
  30. Yang LJ, Wakasa Y, Kawakatsu T, Takaiwa F (2009) The 3′-untranslated region of rice glutelin GluB-1 affects accumulation of heterologous protein in transgenic rice. Biotechnol Lett 31:1625–1631PubMedCrossRefGoogle Scholar
  31. Yasuda H, Tada Y, Hayashi Y, Jomori T, Takaiwa F (2005) Expression of the small peptide GLP-1 in transgenic rice. Transgenic Res 14:677–684PubMedCrossRefGoogle Scholar
  32. Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287:303–305PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Wen Jing Li
    • 1
  • Ling Ling Dai
    • 1
  • Zhi Jian Chai
    • 1
  • Zhi Jie Yin
    • 1
  • Le Qing Qu
    • 1
    Email author
  1. 1.Key Laboratory for Plant Molecular PhysiologyInstitute of Botany, The Chinese Academy of SciencesBeijingChina

Personalised recommendations