A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase Shows High Glyphosate Tolerance in Escherichia coli and Tobacco Plants

Abstract

A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops.

Genetically transformed tobacco plants expressing synthetic EPSPS gene confer tolerance against glyphosate herbicide

“…Thus, the transgenic tobacco lines showing relatively higher transgene expression offered higher enzymatic activity after one week of herbicide application. These results are in accordance with findings of Cao et al (2012) who reported higher shikimate levels after 5 days of glyphosate treatment. After initial surge, the shikimate level begins returning to normal, probably due to maintenance of over-expressed EPSPS protein (Liu et al 2015).…”

Transgenic tobacco simultaneously overexpressing glyphosate N-acetyltransferase and 5-enolpyruvylshikimate-3-phosphate synthase are more resistant to glyphosate than those containing one gene

“…Then the PCR product was ligated to pGEM-T Easy vector (Promega Company) for sequencing. The plasmid p3301-121spAM79 (Cao et al 2012) was used as the donor vector. The AM79 aroA gene was replaced by GAT fragment digested with BamHI and SacI to construct the vector p3301-121spGAT (renamed as pSpGAT).…”

“…The AM79 aroA gene was replaced by GAT fragment digested with BamHI and SacI to construct the vector p3301-121spGAT (renamed as pSpGAT). For constructing non signal peptide GAT plant transformation vector p3301-121GAT (renamed as pGAT), we used the GAT gene to replace the G2 fragment in the plasmid p3301-121G2 with the added BamH I and Sac I sites (Cao et al 2012). …”

“…AM79 EPSPS is a type I EPSPS which was isolated from a glyphosate resistant bacteria strain through the metagenomic method. The transgenic experiment and the analysis of EPSPS enzyme kinetic parameters indicated that AM79 EPSPS could be a good candidate for the development of herbicide-resistant crops (Cao et al 2012). Glyphosate N-acetyltransferase (GAT) can detoxify the herbicide glyphosate by N-acetylation of glyphosate, alleviating the suppression of shikimate pathway.…”

“…EPSPS blocks the suppression of the shikimate pathway by producing more EPSPS or slowing down the binding capacity of glyphosate, whereas GAT detoxifies the glyphosate by N-acetylation. Either AM79 aroA or GAT has been shown to confer transgenic plants with high glyphosate resistance (Castle et al 2004;Cao et al 2012). Pyramiding both genes might be able to make transgenic plants more tolerant to glyphosate, that make it possible that farmers could use higher concentration of glyphosate to kill the tolerant weeds.…”