红掌WRKY家族转录组挖掘及AaWRKY1应答叶斑病的分子机制研究

China has became one of the main producers for Anthurium in the world, but serious devastation from bacterial blight (Xanthomonas axonopodis pv. Dieffenbachiae, Xad) was prevalent in the production. For the scarcity of systemic studies, the molecular pathologic mechanism of this disease remain unknown yet, and no effective measures for blight control with economic and environmental benefit, currently. The plant WRKY superfamily consists of great numbers of transcription factors (TFs) involved in various plant processes. Identification and functional investigation of the mumbers coping with biotic stresses, would enable better exploitation of plant defense responses and facilitate to resistance breeding. .In this study, based on an Anthurium transcriptomic database platform developed in our group previously, a total of 95 candidates with conserved WRKY domains or putative orthologues from other plants, were identified and phylogenetic analysized. For a selected subset of candidate defense-related WRKY TFs, Real-time quantitative RT-PCR were performed to determined its expression in both resistant and in sensitive Anthurium cultivars during Xad infection. So far, 3 blight response WRKY genes has been identified, among which, AaWRKY1 showed a strongly up-regulated expression in resistant Anthurium cultivars, suggesting a putative role in defense response to bacterial blight. .Further studies on AaWRKY1 will demonstrate its structure, origin, expression, and TF regulation activity. Furthermore, we will reveal the defense signaling pathways which AaWRKY1 involved, its roles in Xcd stresses response and utilization potential for blight resistant. This study expands the research field of Anthurium, new results will facilitate to understanding the molecular pathologic mechanism of bacterial blight, also provide theoretical basis for chemical control and molecular breeding for disease resistance in Anthurium.

我国已成为高档花卉红掌的主产地之一，但在生产中普遍遭受细菌性叶斑病的严重危害，其分子病理学机制的缺乏研究认识，目前仍缺少经济环保的高效防治措施。植物WRKY家族是一类有重要调节作用的转录因子，其中病害胁迫相关成员的研究，对阐明病害机制解析和抗病育种有重要意义。本项目拟在前期已完成的转录组测序基础上，系统挖掘出95个红掌WRKY家族Unigenes，通过系统发育分析，评价WRKY基因间的进化关系；结合同源性比较和病原侵染差异表达谱分析，鉴定出病害响应的WRKY成员。进一步研究将阐明病害诱导表达的AaWRKY1基因结构、进化、表达规律、转录调节活性，探讨该基因介导的胁迫信号通路，病害应答机制及其抗病利用潜力。研究将拓展了红掌的基础研究领域，结果有助于解析红掌叶斑病的分子病理学机制，为生产中的化学激素抗病调控，及抗病分子育种提供理论参考。

红掌是广受欢迎的高档盆栽和切花花卉植物，我国已成为世界主要的红掌产地和消费市场；细菌性疫病是红掌生产中的最大威胁，普遍遭受严重危害，目前仍缺少经济环保的有效防治措施。本研究收集鉴定了我国主要红掌栽培区病原菌株，解释了存在广泛的种群遗传多样性和致病力变异，并开发出一套基于LAMP新型检测鉴定方法和试剂盒。建立了可靠的红掌疫病抗性接种鉴定方法，分析了红掌生理特征和疫病抗性水平的相关性，并筛选出具有广谱抗疫病的红掌种质。完成了红掌混合组织转录组测序分析，挖掘出WRKY家族转录因子基因。分别对叶片侵染和系统侵染组织进行RNA seq差异表达分析，筛选分别参与叶片抗性和系统抗性响应的基因表达谱，显示茉莉酸（JA）传导途径在两种抗性的形成中都起重要的作用。筛选出红掌中稳定表达、可用于病害胁迫响应基因实时荧光定量PCR (qRT-PCR)分析的内参基His3、UBQ7；验证出了10个疫病响应相关的WRKY基因，克隆了全长cDNA，并研究其结构特征、系统进化、表达规律、细胞核定位、转录活性等。通过异源表达分析，着重研究了红掌AaWRKY1基因对植物基础防卫防卫PRs的调控作用，过表达增强了植物对病原菌的广谱抗性。研究结果拓展了对红掌细菌性疫病的认识，有助于该病害的综合防治、病理学研究及抗病育种工作的开展。