Hotspot突变介导的油菜EPSPS草甘膦抗性改造与功能验证  

Improving Glyphosate-resistance of EPSPS from Brassica napus Mediated by Hotspot Mutation and Its Function Validation

作　　者：柯达 何云浩 吴方 曾玉兰 华雨 戴成 吴高兵[1] KE Da;HE Yun-Hao;WU Fang;ZENG Yu-Lan;HUA Yu;DAI Cheng;WU Gao-Bing(College of Plant Science and Technology,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]华中农业大学植物科学技术学院,武汉430070

出　　处：《农业生物技术学报》2023年第5期889-900,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31471452);国家转基因生物新品种培育科技重大专项(2018ZX08009-06B)。

摘　　要：草甘膦通过抑制植物5-烯醇式丙酮酰莽草酸-3-磷酸合酶(5-enolpyruvyl shikimate-3-phosphate synthase,EPSPS),无选择性地杀死绝大多数草本植物。通过转基因策略,将对草甘膦具有抗性的EPSPS酶编码基因转入作物,可培育耐草甘膦的作物。当前商业应用的抗草甘膦EPSPS主要来源于微生物,这种“跨界”的基因转移更容易引发人们对转基因安全的顾虑。因此,改造作物自身的EPSPS酶,进而利用内源基因培育耐除草剂作物,具有较大的应用价值。本研究通过构建甘蓝型油菜(Brassica napus)EPSPS突变热点(Hotspot)小型突变体文库,进而筛选高抗草甘膦的突变体。结果显示,获得了5个对除草剂抗性提高的突变体,其中突变体mBnEPSPS表现出最高的草甘膦抗性。相对于野生型BnEPSPS,突变体催化常数kcat由515/s下降到257/s,酶活下降了50%。草甘膦抑制动力学分析表明,存在草甘膦时,mBnEPSPS与BnEPSPS转换数kcat(gly)分别为116/s和13/s,突变体对草甘膦的抗性提高了8.9倍。酶学性质分析表明突变基本不改变酶对植物细胞微环境的适应性。mBnEPSPS转入烟草(Nicotiana benthamiana)后,能够使烟草耐受5倍于商业推荐剂量的农达(41%草甘膦异丙胺盐)处理,这一结果表明mBnEPSPS具有较高的育种应用价值。本研究为通过转基因和基因编辑培育抗除草剂油菜提供了基因资源和理论指导。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.

关 键 词：甘蓝型油菜:草甘膦 抗除草剂 定向进化 5-烯醇式丙酮酰莽草酸-3-磷酸合酶(EPSPS) 

分 类 号：S188[农业科学—农业基础科学]