Plant Molecular Biology Reporter

, Volume 33, Issue 6, pp 1650–1658 | Cite as

Isolation and Functional Analysis of Convolvulus arvensis EPSPS Promoter

  • Zhaofeng Huang
  • Guirong Wang
  • Hongjuan Huang
  • Shouhui Wei
  • Xinxin Zhou
  • Jinyi Chen
  • Jingchao Chen
  • Chaoxian Zhang
Original Paper


A 1142-bp upstream sequence (named CaEPSPS-P, GenBank accession number: KC107822) of the EPSPS gene from Convolvulus arvensis L was obtained by genome walking. The full-length sequence of the CaEPSPS-P and four deletion mutants were fused to the β-glucuronidase (GUS) gene and introduced into Arabidopsis via Agrobacterium-mediated transformation. Histochemical GUS staining of the transgenic plants showed that the CaEPSPS-P could drive GUS expression in the roots, stems, and leaves, but no visible GUS staining was detected in seeds. Further deletion analysis revealed two positive regulatory regions (−900 to −632 and −632 to −418) responsible for the basal activity of the EPSPS promoter. GUS activity assays indicated that GUS expression can be stimulated by light and glyphosate. In addition, a region between −632 and −400 was necessary for light-induced expression, while a region from −900 to −632 was necessary for glyphosate-induced GUS expression. These results suggested that the CaEPSPS-P was modulated by multiple cis-regulatory elements in distinct and complex patterns to regulate transgene expression.


Glyphosate Arabidopsis thaliana Promoter Transgenic plant 



5-enolpyruvylshikimate-3 phosphate synthase


Cetyl trimethyl ammonium bromide


Complementary DNAs


Quantitative reverse transcription polymerase chain reaction




Murashige and Skoog


Wild type





We thank Li Cui from the Chinese Academy of Agriculture Sciences for helpful discussions and critical reading of this manuscript. This work is funded by Special Fund for Agro-scientific Research in the Public Interest (201303022).


  1. An G, Costa MA, Mitra A, Ha SB, Laszlo M (1988) Organ-specific and developmental regulation of the nopaline synthase promoter in transgenic tobacco plants. Plant Physiol 88:547–552PubMedPubMedCentralCrossRefGoogle Scholar
  2. Benfey PN, Chua NH (1990) The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250:959–966PubMedCrossRefGoogle Scholar
  3. Braford MM (1976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  4. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743PubMedCrossRefGoogle Scholar
  5. DeGennaro FP, Weller SC (1984) Differential sensitivity of field bindweed (Convolvulus arvensis) biotypes to glyphosate. Weed Sci 32:472–476Google Scholar
  6. Dolye JJ, Dolye JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
  7. Fang RX, Ferenc N, Shanthi S, NamHai C (1989) Multiple cis regulatory elements for maximal expression of the Cauliflower Mosaic Virus 35S promoter in transgenic plants. Plant Cell 1:141–150PubMedPubMedCentralCrossRefGoogle Scholar
  8. Gaines TA, Zhang W, Wang D, Bukun B, Chisholm ST, Shaner DL, Nissen SJ, Patzoldt WL (2010) Gene amplification confers glyphosate resistance in Amaranthus palmeri. PNAS 107:1029–1034PubMedPubMedCentralCrossRefGoogle Scholar
  9. Gilmartin PM, Sarokin L, Memelink J, Chua NH (1990) Molecular light switches for plant genes. Plant Cell 2:369–378PubMedPubMedCentralCrossRefGoogle Scholar
  10. Goldsbrough PB, Hatch EM, Huang B, Kosinski WG, Dyer WE, Klaus MH, Weller SC (1990) Gene amplification in glyphosate tolerant tobacco cells. Plant Sci 72:53–62CrossRefGoogle Scholar
  11. Gong Y, Liao Z, Chen M, Guo B, Jin H, Sun X, Tang K (2006) Characterization of 5-enolpyruvylshikimate 3-phosphate synthase gene from Camptotheca acuminata. Biol Plant 50(4):542–550CrossRefGoogle Scholar
  12. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res 27:297–300PubMedPubMedCentralCrossRefGoogle Scholar
  13. Huang ZF, Zhang CX, Wei SH, Huang HJ, Liu Y, Cui HL, Chen JC, Yang L, Chen JY (2014) Molecular cloning and characterization of 5-enolpyruvylshikimate-3-phosphate Synthase gene from Convolvulus arvensis L. Mol Biol Rep 41:2077–2084PubMedCrossRefGoogle Scholar
  14. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13):3901–3907PubMedPubMedCentralGoogle Scholar
  15. Jiao Y, Ma L, Strickland E, Deng XW (2005) Conservation and divergence of light-regulated genome expression patterns during seedling development in rice and Arabidopsis. Plant Cell 17:3239–3256PubMedPubMedCentralCrossRefGoogle Scholar
  16. Lam E, Chua NH (1989) ASF-2: A factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in Cab promoters. Plant Cell 1:1147–1156PubMedPubMedCentralCrossRefGoogle Scholar
  17. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van De Peer Y, Rouzé P, Rombauts S (2002) PlantCARE: a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res Database Issue 30:325–327CrossRefGoogle Scholar
  18. Li Y, Sun Y, Yang QC, Kang JM, Zhang TJ, Gruber MY, Fang F (2012) Cloning and function analysis of an alfalfa (Medicago sativa L.) zinc finger protein promoter MsZPP. Mol Biol Rep 39(8):8559–8569PubMedCrossRefGoogle Scholar
  19. Mazarei M, Ying Z, Houtz RL (1998) Functional analysis of the RuBisCo large subunit N-methyltransferase promoter from tobacco and its regulation by light in soybean hairy roots. Plant Cell Rep 17:907–912CrossRefGoogle Scholar
  20. Meilan R, Han KH, Ma C, DiFazio SP, Eaton J (2002) The CP4 transgene provides high levels of tolerance to Roundup® herbicide in field-grown hybrid poplars. Can J Res 32:967–976CrossRefGoogle Scholar
  21. Padgette SR, Kolacz K, Delannay X, Re D, LaVallee B et al (1995) Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci 35:1451–1460CrossRefGoogle Scholar
  22. Priestman MA, Funke T, Singh IM, Crupper SS, Schonbrunn E (2005) 5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate. FEBS Lett 579:728–732PubMedCrossRefGoogle Scholar
  23. Schonbrunn E, Eschenburg S, Shuttleworth WA, Schloss JV, Amrhein N, Evans JN, Kabsch W (2001) Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvyl-shikimate 3-phosphate synthase in atomic detail. Proc Natl Acad Sci U S A 98(4):1376–1380PubMedPubMedCentralCrossRefGoogle Scholar
  24. Shah DM, Horsch RB, Klee HJ, Kishore GM, Winter JA, Tumer NE, Hironaka CM, Sanders PR, Gasser CS, Aykent S, Siegel NR, Rogers SG, Fraley RT (1986) Engineering herbicide tolerance in transgenic plants. Science 233:478–481PubMedCrossRefGoogle Scholar
  25. Steinrucken HC, Amrhein N (1980) The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3-phosphate synthase. Biochem Biophys Res Commun 94:1207–1212PubMedCrossRefGoogle Scholar
  26. Steinrücken HC, Schulz A, Amrhein N, Porter CA, Fraley RT (1986) Overproduction of 5-enolpyruvylshikimate-3-phosphate synthase in a glyphosate-tolerant Petunia hybrida cell line. Arch Biochem Biophys 244:169–178PubMedCrossRefGoogle Scholar
  27. Tian YS, Xu J, Xiong AS, Zhao W, Fu XY, Peng RH, Yao QH (2011) Improvement of glyphosate resistance through concurrent mutations in three amino acids of the ochrobactrum 5-enopyruvylshikimate-3-phosphate synthase. Appl Environ Microbiol 77:8409–8414PubMedPubMedCentralCrossRefGoogle Scholar
  28. Weaver SE, Riley WR (1982) The biology of Canadian weeds. 53. Convolvulus arvensis L. Can J Plant Sci 62:461–472CrossRefGoogle Scholar
  29. Westwood JH, Weller SC (1997) Cellular mechanismsinfluence differential glyphosate sensitivity in field bindweed (Convolvulus arvensis). Weed Sci 45(1):2–11Google Scholar
  30. Westwood JH, Yerkes CN, DeGennaro FP, Weller SC (1997) Absorption and translocation of glyphosate in tolerant and susceptible biotypes of field bindweed (Convolvulus arvensis). Weed Sci 45(4):658–663Google Scholar
  31. Yi Y, Qiao DR, Bai LH, Xu H, Li Y (2007) Cloning, expression, and functional characterization of the Dunaliella salina 5-enolpyruvylshikimate-3-phosphate synthase gene in Escherichia coli. J Microbiol 45(2):153–157PubMedGoogle Scholar
  32. Yu Q, Abdallah I, Han HP, Owen M, Powles S (2009) Distinct non-target site mechanisms endow resistance to glyphosate, ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant Lolium rigidum. Planta 230:713–723PubMedCrossRefGoogle Scholar
  33. Yuan CI, Chaing MY, Chen YM (2002) Triple mechanisms of glyphosate-resistance in a naturally occurring glyphosate-resistant plant Dicliptera chinensis. Plant Sci 163:543–554CrossRefGoogle Scholar
  34. Zhang M, Zhang CX, Fang F, Liu WW, Guo F (2011) Glyphosate tolerance comparison of field bindweed (Convolvulus arvensis) population from different areas. Weed Sci (from China) 29(2):40–42, in ChineseGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Zhaofeng Huang
    • 1
  • Guirong Wang
    • 1
  • Hongjuan Huang
    • 1
  • Shouhui Wei
    • 1
  • Xinxin Zhou
    • 1
  • Jinyi Chen
    • 1
  • Jingchao Chen
    • 1
  • Chaoxian Zhang
    • 1
  1. 1.Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijingChina

Personalised recommendations