辣椒含DWE的WRKY基因功能相关及其在协同应答高温及青枯菌中作用解析

Pepper（Capsicum annuum） is a one of the most important vegetable worldwide and a typical solanaceaes. Heavy loss in pepper production is frequently caused by diseases, which is usually aggravated by high temperature and high humidity. A better understanding of the signal crosstalk between high temperature & high humidity and pathogen response is helpful for the genetic improvement of disease resistance of pepper and other Solanaceae crops. Previously we found that a duouble W-box element,which is present in the promoter of CaWRKY40, a WRKY transcription factor of Capsicum annuum acting as an important regulator in pepper basal thermotolerance and resistance to Ralstonia solanacearum attack, confered largely the synergistic response of CaWRKY40 to high temperature & high humidity and pathogen infection. In addition, the DWE also found in the promoter of 43 members of the pepper WRKY family,implying that these members may constitute a network synergistically regulating pepper’s response to high temperature & high humidi and pathogen infection. Based on this, the common feathures such as W-box binding, and nuclei targeting will be analyzed on the DWE-WKs in the present study. Additionally, by approaches such as realtime PCR, transient expression and VIGS in pepper plants, chromatin immunoprecipitation, ChIP-seq, the functions of these DWE-WKs in pepper’s synergistic response to high temperature & high humidity and pathogen infection, their transcriptonal activation on each other, and their binding to the promoters of each other will be assayed. Based on the results of the project, the regulatory WRKY network on pepper’s synergistic response to high temperature & high humidity and pathogen infection will be established and the crucial nodes in this networks will be identified, which will not only benefit the development of strategy but also can pruovide useful genes for efficient genetic improvement of Solanaceae disease resistance.

土传病害多的典型茄科植物辣椒在高温高湿下发病严重是制约其生产效益的重要限制因素，剖析辣椒高温高湿下抗病机制有利于其抗病遗传改良。项目前期发现DWE在WRKY基因启动子协同应答青枯菌及高温高湿中起关键作用，且发现43个含有DWE的WRKY蛋白（DWE-WKs），暗示它们可能构成辣椒协调耐高温高湿和抗病调控网络。基于此，本项目进一步分析各DWE-WKs亚细胞定位、结合W盒活性，并采用荧光定量PCR分析、在辣椒植株上瞬间超表达、病毒诱导基因沉默、染色质免疫沉淀（ChIP）、ChIP-seq等手段，分析这些DWE-WKs在辣椒应答高温高湿和抗青枯菌中的表达、作用、彼此相互转录激活、与彼此启动子的相互结合等。通过实施本项目，建立DWE-WKs构成的辣椒协同应答高温高湿和青枯菌侵染的WRKY网络，找出其中的关键调控节点，为辣椒等茄科植物高温高湿下抗病遗传改良提供合理技术策略和具有应用价值的基因。

WRKY最大的植物转录因子家族之一，其成员在植物抗病中起重要作用。本项目前期研究发现CaWRKY40含有由两个W盒组成的DWE原件，且发现不同植物的WRKY基因的启动子中均含有DWE原件，可能在应答病原菌侵染中起重要调节作用。本项目在此基础上进一步找出含有DWE的多个辣椒WRKY基因，分析了它们在辣椒应答青枯菌侵染中的作用，并解析了它们与CaWRKY40在表达和功能上的相互关联，取得了以下主要结论：（1）含DWE的CaWRKY17可转录应答青枯菌侵染或高温高湿逆境胁迫处理，在辣椒抗青枯病或耐高温高湿逆境中起正调节作用，且CaWRKY17可在蛋白水平上与CaWRKY40互作，彼此在抗青枯病或耐高温高湿逆境中互为基础且互相促进, 在转录水平上也互相促进，构成正反馈环起着防御反应信号放大的作用；（2）CaWRKY40还可与CaWRKY71互作，且CaWRKY71在青枯菌侵染中诱导上调，且在辣椒抗青枯病中起正调节作用，CaWRKY40会影响CaWRKY71对下游靶基因的结合，并正调节CaWRKY71的转录表达水平；（3）CaWRKY40b通过直接调控包括CaWRKY40在内的防御基因，对辣椒应答青枯菌侵染起负调节作用，且CaWRKY40b能够结合自身启动子并促进自身的转录表达水平；（4）含DWE的CaWRKY22在青枯菌接种的辣椒植株中表达上调，在辣椒抗青枯病中起正调节作用，且发现CaWRKY22可通过与CaWRKY6、CaWRKY27、CaWRKY40和CaWRKY58组成WRKY网络，在辣椒对青枯病的反应中起到正调节作用。这些结果表明DWE-WKs在辣椒耐高温高湿和抗青枯病过程中存在着明显的表达和功能上的关联，这种关联对辣椒耐高温高湿和抗青枯病中起着关键作用。这些结果有助于更加深入阐明WRKY网络介导的辣椒应答青枯菌和高温高湿逆境的交互作用机制。