负调节球孢白僵菌氧化胁迫反应的Bbtmp1基因转录调控子鉴定及其调控作用

It is one of the important mechanisms to producing high level of reactive oxygen species (ROS) for animal and plant cells to preventing fungal pathogen infection. Collectively, fungal pathogen has to mount a counteracting antioxidant response, called oxidative stress responses, to scavenge or degrade ROS for their survival within the host and disease establishment. Thus, the oxidative stress response is a vital process for fungal pathogen to counteract with the host immune system and is involved in virulence. However,the regulatory mechanism of the oxidative stress response in fungal pathogens is still unclear.We found that a transmembrane protein,Bbtmp1,controlled by the Bbhog1 MAPK signal pathway, negatively regulated oxidative stress response and virulence in the insect fungal pathogen Beauveria bassiana.The findings suggested a valuable cue to explore the mechanism of oxidative stress response regulated by Bbhog1 pathway in the fungal pathogen.In this project,we are going to identify the oxidative stress response element in the promoter of Bbtmp1 by reinroducing the gene containing different lengths of promoter into ?Bbtmp1 mutant and detecting the sensitivities of the complementation transformants to oxidative stress. If the sensitivity to oxidative stresses is not complemented to the wild-type extent by reintroducing Bbtmp1 containing a certain length of the promoter,the response element might be in the flanking sequence of the 5'-end of the promoter. Then the yeast one-hybrid strategy will be used to identify the transcription regulator of Bbtmp1 when the fungus is stressed by oxidation. Following charactreization of roles of the transcription regulator in the fungal pathogen,the target genes of the transcription regulator will be identified and characterized using chromatin immunoprecipitation (CHIP) method as well as bioinformatics analysis. One of our goals of the project is to expect to achieve a new breakthrough on understanding the regulatory mechanism of oxidative stress response regulated by Bbhog1 pathway in B. bassiana. Another goal is to hope to provide a theoretical basis for enhancement of oxidation resistance and virulence of the fungal agent using genetic engineering.

产生高水平活性氧是动、植物细胞防御病原真菌侵染的重要机制。相应地，病原真菌在侵染过程中需启动一系列的抗氧化反应（即氧化胁迫反应），清除或抵抗活性氧的伤害。可见，氧化胁迫反应是病原真菌与寄主互作的重要环节，与毒力密切相关。目前，对病原真菌氧化胁迫反应的调控机制尚缺乏清晰的认识。课题组研究发现，一个穿膜蛋白Bbtmp1受Bbhog1信号途径的调控，负调节昆虫病原真菌球孢白僵菌氧化胁迫反应和毒力。该发现为研究Hog1途径调控病原真菌氧化胁迫反应提供了重要线索。本项目拟通过鉴定Bbtmp1基因启动子的氧化胁迫反应"应答"元件，采用酵母单杂交技术获取调控Bbtmp1的转录调控子并研究其生物学功能，进一步利用染色质免疫共沉淀（CHIP）分离鉴定转录调控子调控氧化胁迫反应的靶标基因。项目的开展有望在揭示白僵菌氧化胁迫反应的调控机制方面取得新的突破，并为利用基因工程改良菌株抗逆性和毒力提供新的理论依据。

研究发现，HOG1 MAPK信号途径一个下游基因编码一个穿膜蛋白Bbtmp1 (后命名为Ohmm)，负介导昆虫病原真菌球孢白僵菌氧化胁迫反应和毒力。本项目旨在通过鉴定在氧化胁迫条件下调控Ohmm转录的调控蛋白并解析其功能，为探究HOG1信号途径介导Ohmm调控氧化胁迫反应的机制提供理论支撑。结合生物信息学分析、启动子逐步删除、位点突变及回复互补研究，鉴定了Ohmm基因启动子的氧化胁迫反应“应答”元件c-Myc/c-Myb (GATATC)。利用酵母单杂交筛选与验证，并结合生物信息学分析和转录激活等方法鉴定了4个与“应答”元件互作的转录调控子Naf (BBA_04293)、NirA (BBA_01499)、Fstr (BBA_01981)和Clp (BBA_07528)。在氧化胁迫条件下，Ohmm转录受Naf负调控，不同程度地受NirA、Fstf 和Clp的正调控。其中Naf介导球孢白僵菌生长发育、氧化胁迫反应、细胞表面特性和毒力。破坏基因导致菌株生长速率降低、分生孢子产量下降、萌发延迟、对氧化剂menadione和H2O2的敏感性增强、抗氧化酶活性降低，毒力显著降低。NirA介导分生孢子产生、氧化敏感性和毒力。破坏NirA不影响菌株生长，但导致分生孢子产量降低，对H2O2的耐受性增强，而毒力显著下降。Fstf负介导球孢白僵菌分生孢子产生、活力、氧化胁迫反应和毒力。破坏Fstf引起分生孢子产量显著增加、孢子萌发加快、氧化耐受性提高，并增强了菌株逃避昆虫血腔免疫识别的能力及毒力。Clp影响分生孢子产生、负调节氧化胁迫反应。破坏Clp导致分生孢子产生降低，增强了菌株对氧化剂的耐受性。同时研究发现，Ohmm定位于线粒体，介导线粒体膜电位稳态和活性氧产生，并反馈影响HOG1信号途径，为Ohmm负介导氧化胁迫反应的作用机制提供了新的依据。更有趣的发现是，课题组发现Ohmm参与线粒体铁离子稳态，影响血红素合成、呼吸链完整性和呼吸作用，控制病原菌适应昆虫血腔低氧环境及逃避昆虫血腔免疫识别或攻击，影响菌株“招募”肠道细菌的能力，负介导菌株毒力。项目在揭示HOG1 MAPK信号途径通过介导线粒体功能调控病原真菌毒力的机制方面取得了新的突破，并为后续研究提供了重要线索和理想的研究材料。

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