东北林蛙Toll样受体信号通路对病原微生物胁迫的响应及机制

In recent years, the amphibian populations have a trend of global dramatic decline or disappear. One of the main reason is the outbreak of epidemic diseases, including frogs susceptible pathogenic microorganism addicted to Aeromonas hydrophila(Ah) and Rana grylio virus (RGV) both are important pathogenic agents cause frogs infection, caused a serious threat to the survival of the frogs. This study intends to establish transcriptome database of Rana dybowskii’s skin, spleen and other tissues under the stress of Ah, RGV. By RACE, real-time PCR, immune simulation were combined to explore the Toll-like receptors (TLRs) of Rana dybowskii and the corresponding adapter proteins, regulatory factors and effector molecules, with the present of TLR signaling pathway. In addition, the interaction networks, assembly processes and regulatory mechanisms of these protein machines were studied systematically, in order to explore the response mechanism of frogs TLR signaling pathway, which was directed at the pathogenic microorganisms, as well as the immunization strategy against the susceptible microorganisms. These results will help to realize the immune defense capability of R. dybowskii, associated with the infection of susceptible microorganisms. It can also help us collectthe valuable information of in situ conservation of frogs, and conserve and restore the population of endangered amphibian species efficiently.

近年来，两栖类种群数量呈全球性锐减或消失趋势，其主要原因之一是感染性疾病爆发，其中蛙类易感病原微生物嗜水汽单胞菌（Ah）和虹彩病毒（RGV）是导致蛙类感染的重要致病原，对蛙类的生存造成严重威胁。本研究拟建立Ah、RGV胁迫下的东北林蛙（R.dybowskii）皮肤、脾等组织转录组数据库。通过RACE、定量PCR、免疫共沉淀和分子模拟等技术,在基因、蛋白水平上明确东北林蛙的Toll样受体（TLRs）以及TLRs信号通路中的衔接蛋白、关键调控因子和效应分子，绘制东北林蛙的TLRs信号通路，并对这些蛋白质机器的作用网络、组装过程、调控机制进行系统研究。阐明蛙类TLRs信号通路在病原微生物胁迫时的应答机制，以及蛙类应对易感微生物胁迫时所采取的免疫策略。本研究有助于系统评价东北林蛙应对易感微生物侵袭时机体的免疫防御能力，以期更好地开展蛙类就地保护以及濒危两栖类的种群保护和恢复。

近年来，两栖类种群数量呈全球性锐减或消失趋势，其主要原因之一是感染性疾病爆发。两栖类在长期进化过程中，逐渐建立了天然免疫防御系统以抵御环境病原微生物等有害因子的侵害。Toll 样受体（Toll-like receptors，TLR）信号通路可能在其固有免疫防御中发挥重要作用。本项目建立了嗜水气单胞菌（Aeromonas hydrophila）、蛙虹彩病毒（Ranavirus）胁迫下的东北林蛙（Rana dybowskii）皮肤、脾等组织的转录组数据库，获得了东北林蛙非特异性免疫应答途径Toll样受体信号通路关键分子的全长序列；在基因、蛋白水平上明确了东北林蛙Toll样受体信号通路中关键衔接蛋白、调控因子和效应分子，绘制了东北林蛙Toll样受体信号通路图，并对这些蛋白质机器的作用网络、调控机制进行了研究，初步阐明了蛙类Toll样受体信号通路在应对病原微生物胁迫时的应答机制。研究结果表明，在应对微生物侵袭时，东北林蛙机体可快速启动固有免疫防御应答，其Toll样受体信号通路扮演着非常重要的角色。本研究所得数据有助于更好地开展蛙类就地保护以及濒危两栖类的种群保护和恢复。