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Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants

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Summary

5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), the target of the herbicide glyphosate, catalyzes an essential step in the shikimate pathway common to aromatic amino acid biosynthesis. We have cloned an EPSP synthase gene from Arabidopsis thaliana by hybridization with a petunia cDNA probe. The Arabidopsis gene is highly homologous to the petunia gene within the mature enzyme but is only 23% homologous in the chloroplast transit peptide portion. The Arabidopsis gene contains seven introns in exactly the same positions as those in the petunia gene. The introns are, however, significantly smaller in the Arabidopsis gene. This reduction accounts for the significantly smaller size of the gene as compared to the petunia gene. We have fused the gene to the cauliflower mosaic virus 35 S promoter and reintroduced the chimeric gene into Arabidopsis. The resultant overproduction of EPSPs leads to glyphosate tolerance in transformed callus and plants.

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References

  • Ausubel F, Bahnsen K, Hanson M, Mitchell A, Smith H (1980) Cell and tissue culture of haploid and diploid petunia “Mitchell”. Plant Mol Biol News 1:26–32

    Google Scholar 

  • Baker A, Schatz G (1987) Sequences from a prokaryotic genome or the mouse dihydrofolate reductase gene can restore the import of a truncated precursor protein into yeast mitochondria. Proc Natl Acad Sci USA 84:3117–3121

    Google Scholar 

  • Bennett M, Smith J, Heslop-Harrison J (1982) Nuclear DNA amounts in angiosperms. Proc R Soc Lond 216:179–199

    Google Scholar 

  • Brown I, Feix G, Frendewey D (1986) Accurate in vitro splicing of two pre-mRNA plant introns in a HeLa cell nuclear extract. EMBO J 5:2749–2758

    Google Scholar 

  • Chang C, Meyerowitz E (1986) Molecular cloning and DNA sequence of the Arabidopsis thaliana alcohol dehydrogenase gene. Proc Natl Acad Sci USA 83:1408–1412

    Google Scholar 

  • della-Cioppa G, Bauer C, Klein B, Shah D, Fraley R, Kishore G (1986) Translocation of the precursor of 5-enolpyruvylshikimate-3-phosphate synthase into chloroplasts of higher plants in vitro. Proc Natl Acad Sci USA 83:6873–6877

    Google Scholar 

  • Estelle M, Somerville C (1986) The mutants of Arabidopsis. Trends Genet 2:89–93

    Google Scholar 

  • Frischauf A-M, Lehrach H, Poustka A, Murray N (1983) Lambda replacement vectors carrying polylinker sequences. J Mol Biol 170:827–842

    Google Scholar 

  • Gasser C, Winter J, Hironaka C, Shah D (1987) Structure expression and evolution of the 5-enolpyruvylshikimate-3-phosphate synthase genes of petunia and tomato. J Biol Chem (in press)

  • Kislev N, Rubenstein I (1980) Utility of ethidium bromide in the extraction from whole plants of high molecular weight maize DNA. Plant Physiol 66:1140–1143

    Google Scholar 

  • Koornneef M, van Eden J, Hanhart C, Stam P, Braaksma F, Feenstra W (1983) Linkage map of Arabidopsis thaliana. J Hered 74:265–272

    Google Scholar 

  • Leutwiler L, Hough-Evans B, Meyerowitz E (1984) The DNA of Arabidopsis thaliana. Mol Gen Genet 194:15–23

    Google Scholar 

  • Lloyd A, Barnason A, Rogers S, Byrne M, Fraley R, Horsch R (1986) Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens. Science 234:464–466

    Google Scholar 

  • Meyerowitz E, Pruitt R (1986) Arabidopsis thaliana and plant molecular genetics. Science 229:1214–1218

    Google Scholar 

  • Mousdale D, Coggins J (1985) Subcellular localization of the common shikimate pathway enzymes in Pisum sativum L. Planta 163:241–249

    Google Scholar 

  • Pruitt R, Meyerowitz E (1986) Characterization of the genome of Arabidopsis thaliana. J Mol Biol 187:169–183

    Google Scholar 

  • Rogers S, Klee H, Horsch R, Fraley R (1987) Improved vectors for plant transformation: Expression cassette vectors and new selectable markers. Methods Enzymol, in press

  • Sanger F, Nicklen S, Coulson A (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Shah D, Horsch R, Klee H, Kishore G, Winter J, Tumer N, Hironaka C Sanders P, Gasser C, Aykent S, Siegal N, Rogers S, Fraley R (1986) Engineering herbicide tolerance in transgenic plants. Science 233:478–481

    Google Scholar 

  • Southern E (1975) Detection of specific DNA sequences among fragments separated by gel electrophoresis. J Mol Biol 98:503–517

    Google Scholar 

  • Steinrucken H, Amrhein N (1980) The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate synthase. Biochem Biophys Res Commun 94:1207–1212

    Google Scholar 

  • Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119

    Google Scholar 

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Communicated by R.B. Goldberg

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Klee, H.J., Muskopf, Y.M. & Gasser, C.S. Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants. Mol Gen Genet 210, 437–442 (1987). https://doi.org/10.1007/BF00327194

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  • DOI: https://doi.org/10.1007/BF00327194

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