褐飞虱内共生菌多样性及Arsenophonus介导宿主抵御病原真菌侵染的作用机制研究

The endosymbionts in the brown planthopper (Nilaparvata lugens Stål) is one of key regulators that involved in the regulation of host biological characters, such as growth, reproduction and environmental adaptability. Metarhizium flavoviride, a classic entomopathogenic hyphomycete, has broad spectrum, high virulence and environmental safety. Both of them possess broad application prospects in microbial control of N. lugens. The study sought to explore new biocontrol technology for the brown planthopper using "Endosymbiont–N. lugens–M. flavoviride " system as a model. Firstly, the diversity and dynamics of the endosymbionts in N. lugens was assessed by metagenomic sequencing approach. Secondly, the biological functions of an endosymbiotic bacterium Arsenophonus were elucidated through the genetic methods and molecular biotechnologies. Digital gene expression analysis of the downstream genes that respond to infection by M. flavoviride were detected in Arsenophonus-harboring and Arsenophonus-lacking N. lugens populations. From this analysis, we can summarize the possible molecular mechanism of symbiont-mediated resistence to a fungal entomopathogen in N. lugens and find the suits of genes that directly regulated the fungal virulence. Finally, the recombinant fungal strain with higher virulence was constructed by genetic manipulating the key genes that selected as above. Implementation and completion of this project not only to lay the theoretical basis for the study of the coevolution or mutualism relationship between N. lugens and its endosymbiontes, but also to provide new strategy for developing a strong targeted microbial pesticide products.

褐飞虱内共生菌参与调控宿主生长繁殖以及环境适应性，是褐飞虱生物学特性的重要调控因子。绿僵菌是一种经典的丝孢类生防真菌，具有广谱高毒力和良好的环境相容性，两者均在褐飞虱的微生物防治中具有广阔的应用前景。本项目围绕褐飞虱生物防治新技术的开发为主题，以“共生菌-褐飞虱-绿僵菌”为模型，首先通过宏基因组技术系统分析褐飞虱体内共生菌的多样性，探明共生菌种群结构在褐飞虱不同生长发育时期的动态变化规律；其次运用杂交，数字基因表达谱等遗传学和分子生物学技术全面解析褐飞虱内共生细菌Arsenophonus的生物学功能，阐明其在介导褐飞虱抵御黄绿绿僵菌侵染过程中的作用及其分子机制，并筛选能够提高黄绿绿僵菌致病褐飞虱的关键靶标基因，进而构建毒力显著增强的重组工程菌株。项目的实施和完成不仅为研究褐飞虱与共生菌协同进化，互利共生关系奠定理论基础，也为开发具有强针对性和高毒力的真菌杀虫剂产品提供新的策略。

褐飞虱是我国水稻生产中发生和为害面积最大的害虫，其体内含有大量共生菌，与宿主互利共生；同时在体外自然开放环境中存在诸多病原菌，与褐飞虱形成寄生致死关系，两者是褐飞虱生物防治中两类重要微生物资源。本研究以“共生菌-褐飞虱-绿僵菌”为模型，综合运用遗传学、分子生物学和功能基因组学研究手段，系统分析了褐飞虱不同生长发育阶段和不同致害性种群体内共生菌的多样性及其差异，研究了褐飞虱内共生细菌Arsenophonus在介导褐飞虱抵御黄绿绿僵菌侵染过程中的作用及其分子基础。研究结果表明，基于16S rDNA和ITS扩增子高通量测序分析，褐飞虱(TN1种群)卵期、三龄若虫和羽化48 h的雌雄成虫体内共生菌种类具有较高的丰富度和多样性，但共生细菌的丰富度显著高于共生真菌；两个不同致害性褐飞虱种群体内细菌群落组成和种类多样性存在显著差异，其中TN1种群体内细菌物种丰度明显高于Mudgo种群。qRT-PCR检测表明褐飞虱共生细菌Arsenophonus数量随虫龄的增长而呈递增趋势，在刚羽化成虫中达到顶峰。正反杂交结果表明Arsenophonus 是由母体垂直传递给下一代，亲本含菌与否对子代数量和性比均无显著影响。生测实验显示Arsenophonus能显著提高褐飞虱对黄绿绿僵菌的防御能力。比较转录组分析表明，含菌和缺菌褐飞虱种群受黄绿绿僵菌侵染后，来源于褐飞虱和黄绿绿僵菌的差异表达基因有2205和109个。选取黄绿绿僵菌表达量显著上调的一个差异表达基因Mfsod2(超氧化物歧化酶编码基因)进行功能分析，并通过基因超表达策略构建了一株对褐飞虱毒力显著增强的黄绿绿僵菌重组工程菌株。与野生株相比，遗传重组工程菌株分生孢子的抗氧化能力(甲萘醌耐受力)和耐UV-B辐射能力菌显著提高，且对褐飞虱二龄若虫的毒力增强了27.7%。项目研究结果不仅为研究褐飞虱与共生菌互作机制奠定理论基础，也为开发基于共生/寄生关系调控的褐飞虱生物防治新技术提供科学依据和基因资源。