Transformation of rapeseed by mutated epsps gene fused to chloroplast signal peptide towards enhancing tolerance to glyphosate

Abstract

One of the most effective approaches for weed control is production of glyphosate herbicide tolerant crops. Glyphosate blocks plant growth by inhibiting EPSPS (5- enolpyruvylshikimate-3-phosphate synthase) enzyme which in shikimate pathway for the biosynthesis of aromatic amino acids in plants and cause plant death. Manipulation of epsps gene in order to reduce its affinity for glyphosate is one of the best methods for production of glyphosate-tolerant plants. In the previous studies, site-directed mutagenesis was used to confer two point mutations in E. coli epsps gene in order to convert Glycine96 to Alanine (Gly96Ala) and Alanine 183 to Threonine, (Ala183Thr). In this study, mutated epsps gene was fused to the chloroplast signal peptide from B. napus L. epsps gene. Then, the manipulated Ec-epsps gene was cloned in pBI121 as a plant expression vector; Agrobacterium–mediated transformation was used to deliver the recombinant pBI121 in rapeseed cultivar PF-704591. Molecular analyses were used to confirm the presence and expression of the transgene. Bioassay analysis showed that the amount of glyphosate tolerance in transgenic plants was 2 mM, whereas the non-transformed ones were unable to survive in 0.5 mM glyphosate.

Keywords

• rapeseed
• tolerance to herbicide
• manipulated epsps
• glyphosate
• chloroplast signal peptide