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Transgenic Research

, Volume 13, Issue 2, pp 143–154 | Cite as

Bi-directional Duplex Promoters with Duplicated Enhancers Significantly Increase Transgene Expression in Grape and Tobacco

  • Zhijian T. Li
  • Subramamnian Jayasankar
  • D.J. GrayEmail author
Article

Abstract

Novel bi-directional duplex promoters (BDDP) were constructed by placing two identical core promoters divergently on both upstream and downstream sides of their duplicated enhancer elements. Estimates of promoter function were obtained by creating versions of CaMV 35S and CsVMV BDDPs that contained reporter marker genes encoding β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP) interchangeably linked either to the upstream or downstream core promoters. GUS was used for quantitative analysis of promoter function, whereas, EGFP allowed visual qualitative evaluation. In addition, the GUS and EGFP genes placed in downstream positions were modified by translational fusion with neomycin phosphotransferase (NPTII) to allow simultaneous monitoring of promoter activity and selection of stable transformants. These versions of BDDP were compared with each other and with equivalent unidirectional constructs by evaluating their expression in grape and tobacco. For 35S promoter constructs tested in grape somatic embryos (SE), BDDP exhibited transient GUS expression 206- and 300-fold greater in downstream and upstream configurations, respectively, compared to a unidirectional 35S core promoter. Compared with a unidirectional double enhanced 35S promoter, BDDPs exhibited 0.5- and 3-fold increased GUS expression from downstream and upstream core promoters, respectively. The same differences in expression levels determined quantitatively with GUS were distinguished qualitatively with EGFP. Constructs using CsVMV core promoters yielded results relative to those obtained with 35S promoter. For example, the upstream BDDP CsVMV core promoter provided a 200-fold increase in GUS expression compared to a unidirectional core promoter. However, CsVMV promoter was found to have higher promoter activity than 35S promoter in both BDDP and unidirectional constructs. Incorporation of an additional duplicated enhancer element to BDDPs resulted in increased expression. For example, a 35S BDDP with two divergently arranged duplicated enhancer elements resulted in over a 6-fold increase in GUS expression in stably transformed tobacco plants compared to a BDDP with one duplicated enhancer element. Data demonstrate that BDDP composed of divergently-arranged core promoters separated by duplicated enhancers, all derived from a single promoter sequence, can be used to significantly enhance transgene expression and to direct synchronized expression of multiple transgenes.

bi-directional duplex promoter gene expression grape (Vitis viniferapromoter activity tobacco (Nicotiana tabacumtransgenic plants 

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References

  1. Barfield DG and Pua EC (1991) Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation. Plant Cell Rep 10: 308–314.CrossRefGoogle Scholar
  2. Beck CF and Warren RA (1988) Divergent promoters, a common form of gene organization. Microbiol Rev 52: 318–326.PubMedGoogle Scholar
  3. Bell PJ, Bissinger PH, Evans RJ and Dawes IW (1995) A two-reporter gene system for the analysis of bi-directional transcription from the divergent MAL6T-MAL6S promoter in Saccharomyces cerevisiae. Curr Genet 28: 441–446.CrossRefPubMedGoogle Scholar
  4. Burrow MD, Chlan CA, Sen P and Murai N (1990) High frequency generation of transgenic tobacco plants after modified leaf disk cocultivation with Agrobacterium tumefaciens. Plant Mol Biol Rep 8: 124–139.Google Scholar
  5. Cook PR (1999) The organization of replication and transcription.Science 284: 1790–1795.CrossRefPubMedGoogle Scholar
  6. Datla RS, Hammerlindl JK, Pelcher LE, Crosby WL and Selvaraj G (1991) A bifunctional fusion between β-glucuronidase and neomycin phosphotransferase: a broad-spectrum marker enzyme for plants. Gene 101: 239–246.CrossRefPubMedGoogle Scholar
  7. Dresser DW, Jamin SP, Atkins CJ and Guerrier D (2001) An expressed GNRP-like gene shares a bi-directional promoter with SF3A2 (SAP62) immediately upstream of AMH. Gene 277: 163–173.CrossRefPubMedGoogle Scholar
  8. Driver JA and Kuniyki AH (1984) In vitro propagation of Paradox walnut rootstock. HortScience 19: 507–509.Google Scholar
  9. Dynan WS (1989) Modularity in promoters and enhancers. Cell 58: 1–4.Google Scholar
  10. Eszterhas SK, Bouhassira EE, Martin DIK and Fiering S (2002) Transcriptional interference by independently regulated genes occurs in any relative arrangement of the genes and is influenced by chromosomal integration position. Mol Cell Biol 22: 469–479.CrossRefPubMedGoogle Scholar
  11. Fang RX, Nagy F, Sivasubramaniam S and Chua NH (1989) Multiple cis regulatory elements for maximum expression of the cauliflower mosaic virus 35S promoter in transgenic plants. Plant Cell 1: 141–150.CrossRefPubMedGoogle Scholar
  12. François IEJA, Broekaert WF and Cammue BPA (2002) Different approaches for multi-transgene-stacking in plants. Plant Sci 163: 281–295.CrossRefGoogle Scholar
  13. Fry CJ and Farnham PJ (1999) Context-dependent transcriptional regulation. J Biol Chem 274: 29583–29586.PubMedGoogle Scholar
  14. Gatz C, Katzek J, Prat S and Heyer A (1991) Repression of the CaMV 35S promoter by the octopine synthase enhancer element.FEBS Lett 293: 175–178.CrossRefPubMedGoogle Scholar
  15. Goderis IJWM, De Bolle MFC, François IEJA, Wouters PFJ, Broekaert WF and Cammue BPA (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol Biol 50: 17–27.CrossRefPubMedGoogle Scholar
  16. Goosen N and Van de Putte P (1995) The regulation of transcription initiation by integration host factor. Mol Microbiol 16: 1–7.PubMedGoogle Scholar
  17. Gray DJ (1992) Somatic embryogenesis and plant regeneration from immature zygotic embryos of muscadine grape (Vitis rotundifolia) cultivars. Am J Bot 79: 542–546.Google Scholar
  18. Gray DJ (1995) Somatic embryogenesis in grape. In: Jain SM, Gupta PK and Newton RJ (eds), Somatic Embryogenesis in Woody Plants. Vol. 2 (pp. 191–217) Kluwer Academic Publishers, Dordrecht.Google Scholar
  19. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405.Google Scholar
  20. Kay R, Chan A, Daly M and McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236: 1299–1302.Google Scholar
  21. Keddie JS, Tsiantis M, Piffanelli P, Cella R, Htzopoulos P and Murphy DJ (1994) A seed-specific Brassica napus oleosin promoter interacts with a G-box-specific protein and may be bi-directional. Plant Mol Biol 24: 327–340.CrossRefPubMedGoogle Scholar
  22. Lee TI and Young RA (2000) Transcription of eukaryotic proteincoding genes. Annu Rev Genet 34: 77–137.CrossRefPubMedGoogle Scholar
  23. Lemon B and Tjian R (2000) Orchestrated response: a symphony of transcription factors for gene control. Genes Develop 14: 2551–2569.PubMedGoogle Scholar
  24. Li Z, Jayasankar S and Gray DJ (2001) Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera). Plant Sci 160: 877–887.CrossRefPubMedGoogle Scholar
  25. Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y et al. (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37: 49–59.PubMedGoogle Scholar
  26. Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497.Google Scholar
  27. Peach C and Velten J (1991) Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol Biol 17: 49–60.CrossRefPubMedGoogle Scholar
  28. Pérez-Martín J and De Lorenzo V (1997) Clues and consequences of DNA bending in transcription. Annu Rev Microbiol 51: 593–628.PubMedGoogle Scholar
  29. Poulsen C and Chua NH (1988) Dissection of 5' upstream sequences for selective expression of the Nicotiana plumbaginifolia rbcS-8B gene. Mol Gen Genet 214: 16–23.CrossRefPubMedGoogle Scholar
  30. Ptashne M and Gann A (1997) Transcriptional activation by recruitment.Nature 386: 569–577.CrossRefPubMedGoogle Scholar
  31. Pua EC (2000) Transgenic brown mustard (Brassica juncea). In: Bajaj YPS (ed), Transgenic Crops I. Biotechnology in Agriculture and Forestry. Vol. 46 (pp. 225–242) Springer, Berlin.Google Scholar
  32. Seong GH, Kobatake E, Miura K, Nakazawa A and Aizawa M (2002) Direct atomic force microscopy visualization of integration host factor-induced DNA bending structure of the promoter regulatory region on the Pseudomonas TOL plasmid. Biochem Biophys Res Commun 291: 361–366.PubMedGoogle Scholar
  33. Shigekane H, Kawaguchi Y, Shirakata M, Sakaguchi M and Hirai K (1999) The bi-directional transcriptional promoters for the latency-relating transcripts of the pp38/pp24 mRNAs and the 1.8 kb-mRNA in the long inverted repeats of Marek's disease virus serotype 1 DNA are regulated by common promoterspecific enhancer. Arch Virol 144: 1893–1907.CrossRefPubMedGoogle Scholar
  34. Shlyakhtenko LS, Hsieh P, Grigoriev M, Potaman VN, Sinden RR and Lyubchenko YL (2000) A cruciform structural transition provides a molecular switch for chromosome structure and dynamics. J Mol Biol 296: 1169–1173.CrossRefPubMedGoogle Scholar
  35. Smale ST (2001) Core promoters: active contributors to combinational gene regulation. Genes Develop 15: 2503–2508.PubMedGoogle Scholar
  36. Valerius O, Brendel C, Duvel K and Braus GH (2002) Multiple factors prevent transcriptional interference at the yeast ARO4-HIS7 locus. J Biol Chem 277: 21440–21445.CrossRefPubMedGoogle Scholar
  37. Velten J and Schell J (1985) Selection-expression plasmid vectors for use in genetic transformation of higher plants. Nucleic Acids Res 13: 6981–6997.PubMedGoogle Scholar
  38. Wefald FL, Devlin BN and Williams RS (1990) Functional heterogeneity of mammalian TATA-box sequences revealed by interaction with a cell-specific enhancer. Nature 344: 260–262.CrossRefPubMedGoogle Scholar
  39. West AG, Gaszner M and Felsenfeld G (2002) Insulators: many functions, many mechanisms. Genes Develop 16: 271–288.PubMedGoogle Scholar
  40. Xie M, He Y and Gan S (2001) Bidirectionalization of polar promoters in plants. Nat Biotech 19: 677–679.CrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Zhijian T. Li
    • 1
  • Subramamnian Jayasankar
    • 2
  • D.J. Gray
    • 1
    Email author
  1. 1.Mid-Florida Research and Education Center, Institute of Food and Agricultural SciencesUniversity of FloridaApopkaUSA
  2. 2.Department of Plant Agriculture, Ontario Agricultural CollegeUniversity of GuelphVineland StationCanada

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