摘要
为培育高抗草甘膦作物以应对草甘膦杂草进化,从海洋细菌中筛选到1株高抗草甘膦的盐单胞菌属菌株(Halomonassp.),通过基因组测序及生物信息学分析,确定该菌株的EPSPS基因,在Escherichiacoli(DE3)中对fHoEPSPS、mfHoEPSPS(G384A位点突变)和mHoEPSPS(mfHoEPSPS N端缺失PDT)进行重组表达和纯化,并运用自切割肽LP4/2A介导的基因聚合策略,将抗草铵膦的酶(Repat)置于mHoEPSPS的N端,构建了双抗草甘/铵膦酶(RLH),将其编码基因导入烟草后赋予草甘/铵膦复合抗性。结果显示,该菌株的EPSPS基因(fHoEPSPS)可编码一个N段融合了预苯酸脱水酶(PDT)的双功能酶。草甘膦抗性分析显示mfHoEPSPS的抗性比fHoEPSPS提高了19倍,将mHoEPSPS基因导入烟草后可赋予烟草3倍推荐剂量的草甘膦耐受性,转RLH基因的烟草能够耐受3~5倍推荐剂量的草甘/铵膦复合除草剂。结果表明,源于Halomonas sp.的抗草甘膦EPSPS是一种新型草甘膦耐受酶,通过G384A的位点突变可提高酶活;利用自切割肽介导的基因堆叠策略获得的转RLH基因烟草表现出较高草甘/铵膦复合抗性。
A highly glyphosate resistant strain of Halomonas sp.was screened from marine bacteria to breed crops with high glyphosate resistance to cope with the evolution of glyphosate weeds.The gene encoding EPSPS in this strain was identified through sequencing genome and bioinformatics analysis.The fHoEPSPS,mfHoEPSPS(G384A site mutant),and mHoEPSPS(a mutant with N-end PDT deletion of mfHoEPSPS)were recombinantly expressed and purified in E.coli(DE3).A gene pyramiding strategy mediated by the self-cleaving peptide LP4/2A was used to locate the glufosinate-resistant enzyme(Repat)at the N-end of mHoEPSPS.A dual resistance to glyphosate/ammonium phosphatase(RLH)was constructed.Tobacco transformed with the RLH gene exhibited simultaneous resistance to glyphosate/glufosinate compound herbicides.The results showed that the EPSPS gene(fHoEPSPS)of this strain encoded an N-end fused with a bifunctional enzyme of prephenate dehydratase(PDT).The results of analyzing glyphosate resistance showed that the resistance of mfHoEPSPS was 19 times higher than that of fHoEPSPS.Introducing the coding gene of mHoEPSPS into tobacco endowed tobacco with three times the recommended dose of glyphosate tolerance.Tobacco plants transformed with the RLH gene had simultaneous tolerance 3-5 times the recommended dosage of glyphosate/ammonium phosphine compound herbicides.It is indicated that the glyphosate-resistant EPSPS derived from Halomonas sp.is a new type of glyphosate-tolerant enzyme.The enzyme activity is further improved through the G384A site mutation.The transgenic tobacco with RLH gene obtained through gene pyramiding strategy mediated by the self-cleaving peptides shows high glyphosate/glufosinate compound resistance.It will provide ideas for dealing with the evolution of glyphosate weeds.
作者
丁宁
何云浩
吴琳绯
李婵娟
吴高兵
DING Ning;HE Yunhao;WU Linfei;LI Chanjuan;WU Gaobing(College of Plant Science and Technology,Huazhong Agricultural University,Wuhan 430070,China;College of Food and Biological Engineering,Wuhan Institute of Design and Science,Wuhan 430205,China;College of Life Science and Technology,Huazhong Agricultural University,Wuhan 430070,China)
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2024年第3期158-166,共9页
Journal of Huazhong Agricultural University
基金
国家自然科学基金项目(31971383)
