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

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.

Transformation of canola by chit33 gene towards improving resistance to Sclerotinia sclerotiorum

“…The transgenic lines showed to carry one or two copies of the chi33 gene. The same findings have been reported for transformation of B. napus by Kahrizi et al (2007). Many factors such as the transgene localisation and the copy number (Finnegan & McElroy 1994;Iyer et al 2000;Matzke et al 2000) can contribute to the variation in the transgene expression.…”

Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance

“…Spencer et al, 2000;Howe et al, 2002;Lebrun et al, 2003;Kahrizi et al, 2007;Alibhai et al, 2010). The P106 and then later the TIPS mutation were first found empirically in a mutational screen in Salmonella sp.…”

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase

“…Numerous amino acid residues that affect EPSPS glyphosate resistance have already been identified (1,8,10,13,15,19,20,24,25,31,35,40,50). Notably, these residues are mostly distributed in two regions according to their 3D structure.…”