biologia plantarum

International journal on Plant Life established by Bohumil Němec in 1959

Biologia plantarum 50:542-550, 2006 | DOI: 10.1007/s10535-006-0086-1

Characterization of 5-enolpyruvylshikimate 3-phosphate synthase gene from Camptotheca acuminata

Y. Gong1,3, Z. Liao2, M. Chen2, B. Guo1, H. Jin1, X. Sun2, K. Tang1,2,*
1 Shanghai Key Laboratory of Biotechnology, Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai, China
2 State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China
3 Faculty of Life Science and Biotechnology, Ningbo University, Ningbo, China

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a critical enzyme in the shikimate pathway. The full-length EPSPS cDNA sequence (CaEPSPS, GenBank accession number: AY639815) was cloned and characterized for the first time from woody plant, Camptotheca acuminata, using rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of CaEPSPS was 1778 bp containing a 1557 bp ORF (open reading frame) encoding a polypeptide of 519 amino acids with a calculated molecular mass of 55.6 kDa and an isoelectric point of 8.22. Comparative and bioinformatic analyses revealed that CaEPSPS showed extensive homology with EPSPSs from other plant species. CaEPSPS contained two highly conserved motifs owned by plant and most bacteria EPSPSs in its N-terminal region. Phylogenetic analysis revealed that CaEPSPS belonged to dicotyledonous plant EPSPS group. Tissue expression pattern analysis indicated that CaEPSPS was constitutively expressed in leaves, stems and roots, with the lower expression being found in roots. The coding sequence of CaEPSPS gene was successfully subcloned in a plasmid-Escherichia coli system (pET-32a), and the cells containing the plasmid carrying the CaEPSPS gene exhibited enhanced tolerance to herbicide glyphosate, compared to the control.

Keywords: RACE; shikimate pathway; tissue expression pattern
Subjects: Camptotheca acuminata; dendrogram, phylogenetic tree, phenogram; 5-enolpyruvylshikimate 3-phosphate synthase; polymerase chain reaction (PCR); reverse transcriptase polymerase chain reaction (RT-PCR)

Received: January 22, 2005; Accepted: October 1, 2005; Published: December 1, 2006  Show citation

ACS AIP APA ASA Harvard Chicago Chicago Notes IEEE ISO690 MLA NLM Turabian Vancouver
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. Biologia plantarum, 50(4), 542-550. doi: 10.1007/s10535-006-0086-1
Share...
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)
    • Open full article

    References

    1. Baerson, S.R., Rodriguez, D.J., Tran, M., Feng, Y.M., Biest, N.A., Dill, G.M.: Glyphosate-resistant goosegrass: identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase.-Plant Physiol. 129: 1265-1275, 2002. Go to original source...
    2. Bentley, R.: The shikimate pathway-a metabolic tree with many branches.-Crit. Rev. Biochem. mol. Biol. 25: 307-384, 1990. Go to original source...
    3. Charles, I.J., Keyte, J.W., Brammar, W.J., Smith, M., Hawkins, A.R.: Structure and nucleotide sequence of the complex AROM locus of Aspergillus nidulans.-Nucl. Acids Res. 14: 2201-2213, 1986. Go to original source...
    4. Comber, C., Blanchet, C., Geourjon, C., Deleage, G.: NPS@:Network Protein Sequence Analysis.-Trends biochem. Sci. 25: 147-150, 2000. Go to original source...
    5. Duncan, K., Lewendon, A., Coggins, J.R.: The complete amino acid sequence of Escherichia coli 5-enol-pyruvyl-shikimate-3-phosphate synthase.-FEBS Lett. 170: 59-63, 1984. Go to original source...
    6. Forlanni, G., Parisi, B., Nielsen, E.: 5-enol-pyruvyl-shikimate-3-phosphate synthase from Zea mays cultured cells.-Plant Physiol. 105: 1107-1114, 1994. Go to original source...
    7. Garbe, T., Jones, C., Charles, I., Dougan, G., Young, D.: Cloning and characterization of the aroA gene from Mycobacterium tumberculosis.-J. Bacteriol. 172: 6774-6782, 1990. Go to original source...
    8. Gasser, C.S., Winter, J.A., Hironaka, C.M., Shah, D.M.: Structure, expression and devolution of the 5-enol-pyruvyl-shikimate-3-phosphate synthase genes of Petunia and tomato.-J. biol. Chem. 263: 4280-4287, 1988. Go to original source...
    9. Herrmann, K.M., Weaver, L.M.: The shikimate pathway.-Annu. Rev. Plant Physiol. Plant mol. Biol. 50: 473-503, 1999. Go to original source...
    10. Howe, A.R., Gasser, C.S., Brown, S.M., Padgette, S.R., Hart J., Parker, G.B., Fromm, M.E., Armstrong, C.L.: Glyphosate as a selective agent for the production of fertile transgenic maize (Zea mays L.) plants.-Mol. Breed. 10: 153-164, 2002. Go to original source...
    11. Huynb, Q.K., Kisbore, G.M., Bild, G.S.: 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli: identification of Lys-22 as a potential active site residue.-J. biol. Chem. 263: 735-739, 1988. Go to original source...
    12. Klee, H.J., Muskopf, Y.M., Gasser, C.S.: Cloning of an Arabidopsis thaliana gene encoding 5-enol-pyruvyl-shikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plant.-Mol. gen. Genet. 210: 437-442, 1987. Go to original source...
    13. Kumar, S.K., Tamura, K., Jakobsen, I.B., Nei, M.: MEGA2: molecular evolutionary genetics analysis software.-Bioinformatics 17: 1244-1245, 2001. Go to original source...
    14. Liao, Z., Chen, M., Guo, L., Gong, Y., Tan, F., Sun, X., Tang, K.: Rapid isolation of good-quality total RNA from Taxus and Ginkgo.-Preparative Biochem. Biotechnol. 34: 209-214, 2004. Go to original source...
    15. Marzabadi, M.R., Gruys, K.J., Pansegrau, P.D., Walker, M.C., Yuen, H.K., Sikorski, J.A.: An EPSP synthase inhibitor joining shikimate 3-phosphate with glyphosate: synthesis and ligand binding studies.-Biochemistry 35: 4199-4210, 1996. Go to original source...
    16. McDowell, L.M., Schmidt, A., Cohen, E.R., Studelska, D.R., Schaefer, J.: Structural constraints on the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase from rotational-echo double-resonance NMR.-J. mol. Biol. 256: 160-171, 1996. Go to original source...
    17. Ream, J.E., Steinrcken, H.C., Porter, C.A., Sikorsky, J.A.: Purification and properties of 5-enol-pyruvyl-shikimate-3-phosphate synthase from dark-grown seedlings of Sorghum bicolor.-Plant Physiol. 87: 232-238, 1988. Go to original source...
    18. Saitou, N., Nei, M.: The neighbor-joining method: a new method for reconstructing phylogenetic trees.-Mol. Biol. Evol. 4: 406-425, 1987.
    19. Schonbrunn, E., Eschenburg, S., Shuttleworth, W.A., Schloss, J.V., Amrhein, N., Evans, J.N.S., Kabsch, W.: Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail.-Proc. nat. Acad. Sci. USA 98: 1376-1380, 2001. Go to original source...
    20. Schwede, T., Kopp, J., Guex, N., Peitsch, M.C.: SWISS-MODEL: an automated protein homology-modeling server.-Nucl. Acids Res. 31: 3381-3385, 2003. Go to original source...
    21. Sikorski, J.A., Gruys, K.J.: Understanding glyphosate's molecular mode of action with EPSP synthase: Evidence favoring an allosteric inhibitor model.-Assoc. chem. Res. 30: 2-8, 1997. Go to original source...
    22. Thomas, C.J., Rahier, N.J., Hecht, S.M.: Camptothecin: current perspectives.-Bioorgan. med. Chem. 12: 1585-1604, 2004. Go to original source...
    23. Wang, H.Y., Li, Y.F., Xie, L.X., Xu, P.: Expression of a bacterial aroA-M1, encoding 5-enolpyruvylshikimate-3-phosphate synthase for the production of glyphosate-resistant tobacco plants.-J. Plant Res. 116: 455-460, 2003. Go to original source...
    24. Xu, J.W., Feng, D.J., Li, X.G., Chang, T.J., Zhu, Z.: Cloning of genomic DNA of rice 5-enolpyruvylshikimate-3-phosphate synthase gene and chromosomal localization of the gene.-Sci. China (Ser. C) 45: 251-259, 2002. Go to original source...
    25. Ye, G.N., Hajdukiewicz, P.T., Broyles, D., Rodriguez, D., Xu, C.W., Nehra, N., Staub, J.M.: Plastid-expressed 5-enol-pyruvyl-shikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco.-Plant J. 25: 261-270, 2001. Go to original source...

    Return to the content