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青枯菌铜抗性基因copA的功能
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  • 英文篇名:Function of Copper-Resistant Gene copA of Ralstonia solanacearum
  • 作者:王晓宁 ; 梁欢 ; 王帅 ; 方文生 ; 许景升 ; 冯洁 ; 徐进 ; 曹坳程
  • 英文作者: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;
  • 关键词:青枯菌 ; 铜抗性 ; copA ; 致病性
  • 英文关键词:Ralstonia solanacearum;;copper-resistant;;copA;;pathogenicity
  • 中文刊名:中国农业科学
  • 英文刊名:Scientia Agricultura Sinica
  • 机构:中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室;
  • 出版日期:2019-03-01
  • 出版单位:中国农业科学
  • 年:2019
  • 期:05
  • 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)
  • 语种:中文;
  • 页:66-77
  • 页数:12
  • CN:11-1328/S
  • ISSN:0578-1752
  • 分类号:S432.4
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型菌株下降,接种第10天,copA基因缺失菌株的病情指数较Po82野生型菌株下降了11.7%。copA的缺失导致青枯菌对α-D-葡萄糖和D-海藻糖等碳源,L-丙氨酸和葡萄糖醛酰胺等氮源的代谢利用速率降低,使青枯病发病病程延长。与野生型菌株Po82相比,copA基因缺失菌株中III型分泌系统的转录激活因子编码基因hrpG和hrpB,III型效应子RipX编码基因ripX的表达量显著下调。【结论】铜抗性基因copA在青枯菌铜胁迫应答、致病性等方面发挥一定的作用,研究结果可为进一步解析青枯菌的铜抗性分子机制以及铜抗性菌株的防治提供理论依据。
        【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. The MIC value of copA deletion strain was 0.8 mmol·L~(-1), which decreased by 33.3% compared with that of wild-type strain(1.2 mmol·L~(-1)). The complementary strain restored copper resistance. The results indicated that copA played an important role in copper resistance of R. solanacearum. Compared with wild-type strain, the logarithmic growth rate of copA gene deletion strain decreased in both NA medium and NA medium containing 0.6 mmol·L~(-1) CuSO4, indicating that copA was related to the growth rate of R. solanacearum. The absence of copA resulted in a decrease in the pathogenicity of R. solanacearum. On the 10 th day of inoculation, the disease index of the copA gene deletion strain decreased by 11.7% compared with that of the wild-type strain Po82.The absence of copA resulted in a reduction of metabolic utilization rate of carbon sources such as α-D-glucose, D-trehalose and nitrogen sources such as L-alanine and glucuronide. Compared with wild-type strain Po82, the expression level of hrp B, hrp G and rip X genes, which are important components of the type Ⅲ secretion system, was also significantly down-regulated in copA gene deletion strain. 【Conclusion】 The copper-resistant gene copA plays an important role in copper stress response and pathogenicity of R. solanacearum. The results provide a theoretical basis for further analysis of copper resistance mechanism and the control of copper-resistant strains.
引文
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