Plant Cell Reports

, Volume 26, Issue 1, pp 95–104

Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of E. coli (k12) and transformation of rapeseed (Brassica napus L.) in order to make tolerance to glyphosate

  • Danial Kahrizi
  • Ali Hatef Salmanian
  • Afsoon Afshari
  • Ahmad Moieni
  • Amir Mousavi
Genetics and Genomics

Abstract

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This is a key enzyme in the aromatic amino acid biosynthesis pathway of microorganisms and plants. The manipulation of bacterial EPSPS gene in order to reduce its affinity for glyphosate, followed by its transfer to plants is one of the most effective approaches for the production of glyphosate-tolerant plants. In this study, we chose to focus on amino acid residues glycine96 and alanine183 of the E. coli (k12) EPSPS enzyme. These two amino acids are important residues for glyphosate binding. We used site directed mutagenesis (SDM) to induce point mutations in the E. coli EPSPS gene, in order to convert glycine96 to alanine (Gly96Ala) and alanine183 to threonine (Ala183Thr). After confirming the mutation by sequencing, the altered EPSPS gene was transferred to rapeseed (Brassica napus L.) via Agrobacterium-mediated transformation. The transformed explants were screened in shoot induction medium containing 25 mg L−1 kanamycin. Glyphosate tolerance was assayed in putative transgenic plants. Statistical analysis of data showed that there was a significant difference between the transgenic and control plants. It was observed that transgenic plants were resistant to glyphosate at a concentration of 10 mM whereas the non-transformed control plants were unable to survive 1 mM glyphosate. The presence and copy numbers of the transgene were confirmed with PCR and Southern blotting analysis, respectively.

Keywords

EPSPS Glyphosate resistance Plant transformation Rapeseed (Brassica napus L.) Site-directed mutagenesis 

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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Danial Kahrizi
    • 1
    • 3
  • Ali Hatef Salmanian
    • 2
  • Afsoon Afshari
    • 2
    • 4
  • Ahmad Moieni
    • 1
  • Amir Mousavi
    • 2
  1. 1.Faculty of AgricultureTarbiat Modarres UniversityTehranIran
  2. 2.National Institute for Genetic Engineering and BiotechnologyTehranIran
  3. 3.Faculty of AgricultureRazi UniversityKermanshahIran
  4. 4.Khatam UniversityTehranIran

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