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挖掘草甘膦抗性基因将有助于抗草甘膦遗传改良作物的培育.本研究分离了一株新型草甘膦抗性菌株肠杆菌20 (Enterobacter.E20),该菌株在营养缺陷型培养基中能够耐受高达400 mmol/L草甘膦的胁迫,为充分发掘该菌株在草甘膦抗性方面的利用价值,对该菌株进行了全基因组测序(GenBank:CP012999.1).分离克隆了肠杆菌20其EPSPS (5-烯醇式丙酮酰莽草酸-3-磷酸合酶)编码基因aroA20 (WP_039261572.1),该基因序列长度为1 284 bp,编码427个氨基酸,序列比对分析显示AroA20具有Ⅰ型EPSPS典型的保守结构特征,系统进化分析显示AroA20与大肠杆菌K12的EPSPS具有最近的亲缘关系.利用原核表达系统鉴定了重组aroA 20菌株在草甘膦胁迫下的耐受性,在转基因烟草中过表达了aroA 20基因并鉴定了转基因植株的草甘膦抗性.研究肠杆菌20与其草甘膦的靶标EPSPS编码基因aroA 20在草甘膦抗性上存在差异的分子机制将为深入理解生物体草甘膦抗性奠定研究基础.“,”Excavating glyphosate resistance genes will contribute to the cultivation of glyphosate-tolerant crops.In this study,a novel glyphosate-resistant strain Enterobacter 20 (Enterobacter.E20) was isolated,which was able to tolerate up to 400 mmol/L glyphosate in auxotrophic medium.For exploiting the utilization value of the strain in glyphosate resistance,the whole genome sequencing for Enterobacter.E20 was performed under the GenBank number CP012999.1.aroA 20,the key enzyme in shikimic acid pathway was isolated (WP_039261572.1).The full length of aroA 20 gene was 1 284 bp with encoding 427 amino acids.Sequence alignment analysis showed that AroA20 had type Ⅰ EPSPS typical conserved structural features,and phylogenetic analysis showed that aroA 20 and E.coli K12 EPSPS possessed the closest genetic relationship.The tolerance of recombinant strain harboring aroA 20 under glyphosate stress was identified by prokaryotic expression system.Over-expression of the aroA 20 gene in transgenic tobacco were also be implemented for evaluation of glyphosate resistance.The special molecular mechanism difference between aroA 20 and Enterobacter.E20 might be worthful in further understanding for the resistance of glyphosate in the organisms.