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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

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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.

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Acknowledgments

Special thanks to Dr. P. Shariati and Dr. Christopher Preston for critical reading of this manuscript. The National Institute for Genetic Engineering and Biotechnology (NIGEB), Iran, has financially supported this research project no. 155.

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Authors and Affiliations

  1. Faculty of Agriculture, Tarbiat Modarres University, Tehran, Iran

    Danial Kahrizi & Ahmad Moieni

  2. National Institute for Genetic Engineering and Biotechnology, P.O. Box 14155-6343, Tehran, Iran

    Ali Hatef Salmanian, Afsoon Afshari & Amir Mousavi

  3. Faculty of Agriculture, Razi University, Kermanshah, Iran

    Danial Kahrizi

  4. Khatam University, Tehran, Iran

    Afsoon Afshari

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  1. Danial Kahrizi
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  2. Ali Hatef Salmanian
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  3. Afsoon Afshari
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  4. Ahmad Moieni
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  5. Amir Mousavi
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Correspondence to Ali Hatef Salmanian.

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Communicated by R. Schmidt

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Kahrizi, D., Salmanian, A.H., Afshari, A. et al. 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. Plant Cell Rep 26, 95–104 (2007). https://doi.org/10.1007/s00299-006-0208-4

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  • DOI: https://doi.org/10.1007/s00299-006-0208-4

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