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青枯菌铜抗性基因copA的功能 预览

Function of Copper-Resistant Gene copA of Ralstonia solanacearum
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摘要 【目的】植物细菌性青枯病(bacterial wilt of plants)是由茄科雷尔氏菌(Ralstonia solanacearum)引起的一种世界性重大土传病害。作为防治青枯病等细菌性病害的重要杀菌剂,铜制剂的广泛使用造成多种植物病原细菌群体中出现了铜抗性菌株。青枯菌Po82菌株大质粒上携带了丁香假单胞菌中的铜抗性编码基因copA的同源物,论文旨在探明青枯菌Po82菌株copA在铜抗性、致病性等方面的生物学功能。【方法】以青枯菌Po82菌株为研究对象,利用MEGA6.0软件包,基于邻接法构建copA的系统发育树,探究铜抗性基因copA在青枯菌和其他植物病原细菌中的系统进化关系。通过反向遗传学的研究手段,采用基因同源重组双交换和电击转化法,构建copA基因缺失菌株及相应互补菌株。通过最小抑制浓度(minimal inhibition concentration,MIC)测定、RT-qPCR、Biolog代谢芯片以及致病力等基础生物学测定研究手段,解析copA与青枯菌铜胁迫应答、代谢活性、致病性和运动性等表型生物学特征之间的关系。【结果】同源性比对分析结果显示,copA广泛存在于青枯菌群体中,青枯菌copA在亲缘关系上与耐金属贪铜菌最为紧密,与稻黄单胞菌、丁香假单胞菌和大肠杆菌的亲缘关系较远。RT-qPCR结果显示copA的表达受到铜离子的诱导,copA的表达量随着CuSO4浓度的增加而增加。CuSO4浓度为1.0mmol·L-1时,基因表达量最高。铜MIC测定结果显示copA基因缺失菌株对铜离子的敏感性增加,copA基因缺失菌株的MIC值为0.8 mmol·L-1,较野生型菌株的1.2 mmol·L-1下降了33.3%,互补菌株恢复了铜抗性能力,表明copA在青枯菌的铜胁迫应答过程中发挥着重要作用。与野生型菌株相比,copA基因缺失菌株在普通NA培养基及含0.6mmol·L-1 CuSO4的NA培养基中的对数生长期的生长速率降低,表明copA与青枯菌的生长速率相关。copA基因缺失菌株于发病前期病情指数较野生� 【Objective】 Bacterial wilt of plants, caused by Ralstonia solanacearum, is a major soil-borne disease around the world. As an important bactericide to control bacterial diseases such as bacterial wilt, the widespread use of copper-based bactericides has led to the emergence of copper-resistant strains in a variety of plant pathogenic bacterial population. The copper-resistant coding gene copA, homologous with Pseudomonas syringae, was carried on the megaplasmid of R. solanacearum Po82 strain. The objective of this study is to investigate the biological function of copA in copper resistance and pathogenicity of Po82 strain.【Method】The phylogenetic relationship of the copper-resistant gene copA in different strains of R. solanacearum and other phytobacterial strains was analyzed based on neighbor-joining method using MEGA6.0 for constructing the phylogenetic tree of copA. By means of reverse genetics strategy, using the methods of gene homologous recombination and electroporation, copA gene deletion and complementary strains of Po82 were constructed. Copper minimal inhibition concentration(MIC) test, RT-qPCR,Biolog chip analysis, pathogenicity test and other basic biological methods were employed to clarify the relationship between copA and biological characteristics such as response to copper stress, metabolic activity, pathogenicity, and motility of R. solanacearum.【Result】The results of homology analysis showed that the copA existed widely in the bacterial population, and the copA of R.solanacearum was most closely related to Cupriavidus metallidurans, but far genetic relationship with Xanthomonas oryzae, P.syringae and Escherichia coli. RT-qPCR analysis showed that the expression of copA was induced by copper. The expression of copA increased with the increase of CuSO4 concentration. The expression level of copA was the highest when the Cu SO4 concentration was 1.0 mmol·L-1. By MIC analysis, the result showed that the sensitivity of the copA deletion strain to copper was significantly increased. Th
作者 王晓宁 梁欢 王帅 方文生 许景升 冯洁 徐进 曹坳程 WANG XiaoNing;LIANG Huan;WANG Shuai;FANG WenSheng;XU JingSheng;FENG Jie;XU Jin;CAO AoCheng(State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193)
出处 《中国农业科学》 CAS CSCD 北大核心 2019年第5期837-848,共12页 Scientia Agricultura Sinica
基金 国家重点研发计划(2017YFD0201600,2018YFD0200802) 国家科技支撑计划(2015BAD08B03) 国家自然科学基金(31571975) 中国农业科学院创新工程(CAAS-XTCX2016012).
关键词 青枯菌 铜抗性 COPA 致病性 Ralstonia solanacearum copper-resistant copA pathogenicity
作者简介 王晓宁,E-mail:18811012063@163.com;通信作者:徐进,E-mail:jinxu@ippcaas.cn;通信作者:曹坳程,E-mail:caoac@vip.sina.com
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