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| Improving Glyphosate-resistance of EPSPS from Brassica napus Mediated by Hotspot Mutation and Its Function Validation |
| KE Da, HE Yun-Hao, WU Fang, ZENG Yu-Lan, HUA Yu, DAI Cheng, WU Gao-Bing* |
| 1 College of Plant Science, Technology, Huazhong Agricultural University, Wuhan 430070, China |
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Abstract Glyphosate can kill most herbaceous plants by inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity. Glyphosate-resistant (GR) crops can be developed by transforming the EPSPS gene encoding the glyphosate-resistant enzyme into crops. The current commercial application of GR EPSPS is mainly derived from microorganisms, and such "cross-kingdom" gene transfer is more likely to cause people's concerns about the safety of transgenic food. Therefore, improving the EPSPS of crops and then developing GR crops using crop endogenous genes is a desirable strategy to solve this problem. In this study, the Hotspots mutant library of EPSPS of Brassica napus was constructed, and the mutants resistant to high glyphosate levels were screened. 5 mutants with improved herbicide resistance were obtained, among which mBnEPSPS showed the highest glyphosate resistance. Compared with the wild type (BnEPSPS), the kcat of the mutant decreased from 515/s to 257/s, with a decrease of 50%. In the presence of glyphosate, the turnover numbers kcat (gly) of mBnEPSPS and BnEPSPS were 116/s and 13/s, respectively, meaning an 8.9-folds increase in glyphosate resistance. The mutation did not change the adaptation of enzymes to the plant cell microenvironment. After being transformed to tobacco (Nicotiana benthamiana), mBnEPSPS could confer tobacco resistance to roundup at 5 times higher than the commercial recommended dose of Roundup (41% glyphosate-isopropylammonium), suggesting that mBnEPSPS had a high breeding application value. This study provides genetic resources and a theoretical basis for developing herbicide-resistant rape seed through transgenic and gene editing.
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Received: 15 June 2022
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Corresponding Authors:
*wgb@mail.hzau.edu.cn
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