Document Type: Research Paper
Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
Agricultural Biotechnology Dept.
Agricultural Biotechnology, NIGEB, Tehran, Iran
Department of Biology, Faculty of Science, Lorestan University, Khoramabad, Iran.
Background: Glyphosate is a non-selective systemic herbicide with a broad spectrum of weed control that inhibits a key enzyme, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, in the shikimate pathway.
Objectives: Isolation and analysis of the epsps (aroA) gene responsible for glyphosate-tolerance in bacteria from Roundup-contaminated soils was the aim of this study.
Materials and Methods: Sampling was done from the soil of the gardens which were heavily contaminated by Roundup herbicide and then bacterial screening was performed in the presence of high concentrations of glyphosate. The genus of bacterium was identified via molecular methods such as 16S rRNA sequencing. The aroA gene of this bacterium (aroAHA-09) was isolated using the primers designed-upon specific regions of aroA genes available in NCBI GenBank database. The PCR product was cloned, sequenced and subcloned into pET28a as an expression vector and transferred into E. coli strain BL21(DE3). The cells were inoculated in liquid M9 minimal medium containing IPTG and different concentrations of glyphosate.
Results: The genus of bacterium was identified as Pseudomonas sp. strain HA-09. The isolated aroAHA-09 gene from this bacterium was approximately 2.2 kb in size. Bioassay of E. coli expressing this gene showed high tolerance to glyphosate (up to 300 mM).
Conclusions: The aroAHA-09 gene could be considered as a novel and efficient candidate for development of glyphosate-tolerant crop plants.