青枯菌铜抗性基因簇的表达调控及其功能研究

Bacterial wilt of plants, caused by Ralstonia solanacearum, is one of the most destructive diseases worldwide. Copper compounds have been widely used as bactericides in agroecological systems to control plant diseases, and led to emergence of copper resistance strains in several plant related bacterial species. Up to now, copper resistance of R. solanacearum has not yet been reported. In our preliminary study, the complete genome of R. solanacearum strain Po82, a pathogenic variation, was fully sequenced and annotated. The genome of Po82 is comprised of two circular replicons, a chromosome and a megaplasmid which harbors a putative copper-resistance gene cluster, copSRABCD. Comparative genomic analysis revealed that four out of another five sequenced R. solanacearum strains contain this gene cluster. copSRABCD has high homology with copABCDRS, the well-studied copper resistance system in P. syringae. However, the biological function of the copSRABCD gene cluster has not been characterized in R. solanacearum. The objective of this project is: (1) to unravel the potential role of copSRABCD gene cluster in biological characteristics (e.g. copper resistance, pathogenicity, motility, colonization etc.), the whole copSRABCD gene cluster and each of the six cop genes will be deleted in R. solanacearum strain Po82; (2)to determine the molecular regulatory mechanism of copSRABCD gene cluster, expression of cop genes, identification of transcription unit and interaction between CopR and the promoter of copABCDS were analysed at the transcriptomic levels by Real-time quantitative PCR，RT-PCR and Gel Retardation Assay; (3)to identify the conserved and strain-specific traits required to resist copper stress and wilt plants, high-throughput transcriptome sequencing technology will be used to measure expression of the R. solanacearum Po82 and GMI1000 genomes in planta and in copper-induced culture. This project will be helpful not only to elucidate the molecular adaptation of R. solanacarum to enviromental niches and host plants, but to develop environmentally friendly and economically viable stradegy for bacterial diease control as well.

青枯病是由茄科雷尔氏菌（简称青枯菌）引起的一种世界性重大细菌病害。作为植物细菌性病害的主要防控措施，铜制剂长期大量的使用导致多种植物病原细菌中出现铜抗性菌株。迄今为止，尚未见青枯菌铜抗性及其分子机理相关领域的研究报道。本研究基于青枯菌Po82菌株的全基因组序列信息，以位于其大质粒上可能的铜抗性编码基因簇—copSRABCD为研究对象，采用单个基因和整体敲除策略，以期明确该基因簇及单个基因与青枯菌铜抗性、致病性等表型生物学特征之间的关系；采用RT-PCR、RT-qPCR和凝胶阻滞分析等研究手段，探究青枯菌cop编码系统转录表达的分子调控机理；应用高通量转录组测序技术，比较Po82与GMI1000菌株在营养丰富培养条件、铜诱导培养以及寄主体内环境下共表达及差异表达的基因，以期发掘新的铜抗性及致病相关基因，为进一步深入解析青枯菌寄主及环境适应性进化的分子机理奠定基础。

青枯病（bacterial wilt of plants）是由雷尔氏菌属植物致病种（Ralstonia spp. 泛称青枯菌）引起的一种世界性分布的重大土传病害。铜制剂是防治青枯病等细菌性病害的重要杀菌剂，由于广泛和高频次的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菌株上携带了丁香假单胞菌中的铜抗性编码基因簇copSRABCD的同源物。本研究以该基因簇为研究对象，采用单个基因和整体敲除策略，以期明确该基因簇与青枯菌铜抗性、致病性等表型生物学特征之间的关系；阐明青枯菌铜胁迫应答的分子机制、发掘青枯菌中新的铜抗性和致病相关基因。.通过最小抑制浓度与核酸检测手段探明了铜抗性编码基因簇copABCD普遍存在于青枯菌群体中，但不同青枯菌类群的铜抗性水平差异明显。通过反向遗传学的研究手段，分别构建copABCD、copA、copB、copC和copD基因的缺失菌株及相应互补菌株。揭示出copABCD基因簇及各单基因的缺失造成青枯菌Po82菌株的铜抗性、致病力、生长速率和运动性等生物学功能受到不同程度的损伤。copSR和copABCD均为单一转录单位，copSR和copABCD突变菌株的MIC值相同，均较野生型菌株下降了33.3%。离体培养条件下，亚抑制铜浓度水平诱导不仅可正调控Po82和Po82△copABCD菌株铜抗性相关基因的表达，相当数量的致病相关基因的表达也显著上调。上调表达的致病相关基因大部分为hrp基因簇的组成基因和III型效应子（T3Es）编码基因。在寄主体内环境中，copABCD基因簇缺失菌株上调表达Ⅳ型菌毛编码基因及致病相关基因。