Site-specific Mutagenesis of the Arabidopsis Gene 5-enolpyruvy-shikimate-3-phosphate Synthase (EPSPS) to Gain Glyphosate-resistant Transgenic Arabidopsis thaliana
Abstract：At present, the most widely used gene in commercialized transgenic glyphosate-resistant crops is 5-enolpyruvy-shikimate-3-phosphate synthase (EPSPS) genes from Agrobacterium tumefaciens CP4 and Escherichia coli, which results in narrow gene resource. In this study, aiming at developing new genes, Arabidopsis EPSPS gene was cloned and two amino acids (T178I, P182S) were point mutated via overlap extension PCR. The wild-type gene (AtEPSPS) and the mutant gene (AtTIPS) were introduced into EPSPS-defective strain E. coli ER2799, respectively. The glyphosate resistance of AtEPSPS and AtTIPS transformed ER2799 strains were tested at 0, 5, 10, 20, 50, 100 and 150 mmol/L glyphosate. AtTIPS transformed ER2799 grew normally in the medium containing 20 mmol/L glyphosate, while the growth of AtEPSPS transformed ER2799 was inhibited by 5 mmol/L glyphosate(P＜0.01). When the concentration of glyphosate was higher than 50 mmol/L, the growth of AtTIPS transformed ER2799 was also inhibited, without difference with AtEPSPS transformed ER2799. Meanwhile, we constructed two plant expression vectors containing AtEPSPS or AtTIPS, and introduced them into wild Arabidopsis thaliana via Agrobacterium mediated flower dipping approach, respectively. Seven AtEPSPS transformed lines and ten AtTIPS transformed lines were gained. PCR and RT-PCR analysis suggested that the AtEPSPS and AtTIPS genes were successfully integrated into the Arabidopsis thaliana genome and expressed correctly. We transplanted the transgenic plants to MS media containing 0.3, 0.5 and 1.0 mmol/L glyphosate, respectively, to test their resistance to glyphosate. All three glyphosate concentration led the leaves of AtEPSPS transformed plants and wild type plants to turn yellow, whereas AtTIPS transformed plants grew normally. RT-PCR results showed that there was no significant difference of transcriptional level of AtEPSPS and AtTIPS in transgenic plants. The survival rate and fresh weight of AtTIPS transformed plants were significantly higher than that of AtEPSPS transformed plants (P＜0.05). Whereas, the survival rate and fresh weight of AtEPSPS transformed plants were higher than that of wild type plants. These results suggest that the overexpression of AtEPSPS could only confer transgenic plants with low level of glyphosate tolerance; however, transgenic plants overexpressing AtTIPS could tolerate high concentration of glyphosate. So we can conclude that AtTIPS transformed plants performed higher glyphosate resistance than that of AtEPSPS transformed plants and wild type plants, and AtTIPS can be a candidate for the development of transgenic glyphosate-resistant crops.
