Abstract
The relationship among motif copy number, its distance from the TATA box and expression level was analyzed in transformed tobacco plants. Single or multiple copies of octopine synthase (ocs) enhancer elements from the ocs promoter were linked to the minimal ocs promoter and a β-glucuronidase (GUS) reporter gene, then transformed stably into tobacco. Reporter gene assays revealed that mere repetition of the ocs enhancer sequence is not sufficient for promoter activity. Increasing the number of copies of the ocs element elevated the level of gene expression in an additive manner that was dependent of the element’s distance from the TATA box. To our knowledge, this is the first report in which the regulation of transgene expression by interactions between these two factors has been documented.
Similar content being viewed by others
Abbreviations
- GUS:
-
β-Glucuronidase
- ocs:
-
Octopine synthase
- DR:
-
Directed repeat
- IR:
-
Inverted repeat
References
Bhullar S, Chakravarthy S, Advani S et al (2003) Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping. Plant Physiol 132:988–998. doi:10.1104/pp.103.020602
Bouchez D, Tokuhisa JG, Liewellyn DJ et al (1989) The ocs-element is a component of the promoters of several T-DNA and plant viral genes. EMBO J 8(13):4197–4204
Bradford MH (1976) Rapid and sensitive methods for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3
Cazzonelli CI, Velten J (2008) In vivo characterization of plant promoter element interaction using synthetic promoters. Transgenic Res 17:437–457. doi:10.1007/s11248-007-9117-8
Cazzonelli CI, Burke J, Velten J (2005) Functional characterization of the geminiviral conserved late element (CLE) in uninfected tobacco. Plant Mol Biol 58:465–481. doi:10.1007/s11103-005-6589-x
Chaturvedi CP, Sawant SV, Kiran K et al (2006) Analysis of polarity in the expression from a multifactorial bidirectional promoter designed for high-level expression of transgenes in plants. J Biotechnol 123:1–12. doi:10.1016/j.jbiotec.2005.10.014
Ellis JG, Llewellyn DJ, Walker JC et al (1987) The ocs element: a 16 base pair palindrome essential for activity of the octopine synthase enhancer. EMBO J 6:3203–3208
Fang RX, Nagy F, Sivasubramaniam S et al (1989) Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants. Plant Cell 1:141–150
Geisler M, Kleczkowski LA, Karpinski S (2006) A universal algorithm for genome-wide in silicio identification of biologically significant gene promoter putative cis-regulatory-elements; identification of new elements for reactive oxygen species and sucrose signaling in Arabidopsis. Plant J 45(3):384–398. doi:10.1111/j.1365-313X.2005.02634.x
Horsch RB, Fry JE, Hoffmann NL et al (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231. doi:10.1126/science.227.4691.1229
Ishige F, Takaichi M, Foster R et al (1999) A G-box motif (GCCACGTGCC) tetramer confers high-level constitutive expression in dicot and monocot plants. Plant J 18(4):443–448. doi:10.1046/j.1365-313X.1999.00456.x
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:287–305. doi:10.1007/BF02667740
Koncz C, De Greve H, André D et al (1983) The opine synthase genes carried by Ti plasmids contain all signals necessary for expression in plants. EMBO J 2(9):1597–1603
Leisner SM, Gelvin SB (1988) Structure of the octopine synthase upstream activator sequence. Proc Natl Acad Sci USA 85:2553–2557. doi:10.1073/pnas.85.8.2553
Ni M, Cui DC, Einstein J et al (1995) Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes. Plant J 7:661–676. doi:10.1046/j.1365-313X.1995.7040661.x
Rushton P, Reinstadler A, Lipka V et al (2002) Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signaling. Plant Cell 14:749–762. doi:10.1105/tpc.010412
Sawant SV, Kiran K, Mehrotra R et al (2005) A variety of synergistic and antagonistic interactions mediated by cis-acting DNA motifs regulate gene expression in plant cells and modulate stability of the transcription complex formed on a basal promoter. J Exp Bot 56:2345–2353. doi:10.1093/jxb/eri227
Singh KB, Foley RC, Oñate-Sánchez L (2002) Transcription factors in plant defense and stress responses. Curr Opin Plant Biol 5:430–436. doi:10.1016/S1369-5266(02)00289-3
Venter M (2007) Synthetic promoters: genetic control through cis engineering. Trends Plant Sci 12:118–124. doi:10.1016/j.tplants.2007.01.002
Xie MT, He YH, Gan SS (2001) Bidirectionalization of polar promoters in plants. Nat Biotechnol 19:677–679. doi:10.1038/90296
Acknowledgments
We thank Dr. Decai Cui for providing the construct 8. This research was supported by grants from the National Special Program for Research and Industrialization of Transgenic Plants (Grant No. J99-A-038) and the National Natural Science Foundation of China (Grant No. 30770145).
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by L. A. Kleczkowski.
Rights and permissions
About this article
Cite this article
Liu, S., Bao, Y. Effects of copy number of an octopine synthase enhancer element and its distance from the TATA box on heterologous expression in transgenic tobacco. Acta Physiol Plant 31, 705–710 (2009). https://doi.org/10.1007/s11738-009-0282-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11738-009-0282-7