两种蜜蜂幼虫的球囊菌抗性差异机制研究

Chalkbrood is caused by Ascosphaera apis, a fungal pathogen specially infects honeybee larvae. Chalkbrood is a chronic disease that can result in a sharp decline for honeybee colony population. Apis mellifera ligustica larvae is susceptible to A. apis, while Apis cerana cerana larvae has a strong A. apis-resistance. Currently, due to a lack of in-depth study on honeybee larvae's molecular mechanisms regulating immune responses and regulation networks, none efficient control strategy of chalkbrood is available. The proposer previously used RNA-seq to sequence western honeybee and eastern honeybee larval guts, assembled and annotated the transcriptomes of A. apis and A. c. cerana larval gut, and uncovered the molecular mechanisms underlying the hosts' immune responses to A. apis via trend analysis of differentially expressed genes (DEGs). This project is designed to deep sequence the larval guts of A. m. ligustica and A. c. cerana challenged by A. apis using small RNA-seq technology, detect the expression profiles of microRNA (miRNA), and establish the dfferentially expressed miRNA-dfferentially expressed mRNA (DEmiRNA-DEmRNA) regulatory networks through trend analysis as well as target analysis, and finally, validate the role of key regulatory DEmiRNA (DEmiR-Key) in the larvae's A. apis-resistance by means of overexpression and konckdown of DEmiR-Key. This project aims to elucidate the molecular mechanisms regulating the A. apis-resistance difference between A. m. ligustica larvae and A. c. cerana larvae, and provide potential targets for the treatment of chalkbrood.

白垩病是由球囊菌侵染蜜蜂幼虫而引起死亡的真菌病，是养蜂生产的一大顽疾。意大利蜜蜂(意蜂)幼虫极易被球囊菌侵染而患病，但中华蜜蜂(中蜂)幼虫具有较强的球囊菌抗性。目前，对蜜蜂幼虫在球囊菌胁迫过程中的免疫应答及分子调控机制缺乏分子水平的深入研究，至今仍无防治白垩病的有效方法。申请人前期已利用RNA-seq技术对健康及球囊菌胁迫的两种蜜蜂幼虫肠道进行测序，组装并注释了球囊菌及中蜂幼虫肠道参考转录组，并通过趋势分析揭示了宿主响应胁迫的免疫应答机制。本项目拟利用small RNA-seq技术对球囊菌胁迫的意蜂及中蜂幼虫肠道进行测序，检测宿主的microRNA表达谱，结合趋势分析与靶向分析建立宿主的DEmiRNA-DEmRNA调控网络，并通过过表达和敲减关键miRNA验证其在宿主的球囊菌抗性方面的作用。本项目旨在解析意蜂及中蜂幼虫的球囊菌抗性差异产生的分子机制，并为白垩病治疗提供潜在分子靶点。

白垩病是由蜜蜂球囊菌（球囊菌）侵染蜜蜂幼虫而引起死亡的真菌病，可导致成年蜜蜂数量和蜂群群势的急剧下降，给养蜂生产造成较大损失。意大利蜜蜂（意蜂）和中华蜜蜂（中蜂）是我国养蜂生产中使用的两个主要蜂种。意蜂幼虫极易被球囊菌侵染而患病，但中蜂幼虫具有较强的球囊菌抗性。微小RNA（miRNA）是基因表达调控的关键因子，广泛参与细胞分化、生长发育和免疫防御等生物学过程。本项目通过形态学观察和分子生物学验证证实球囊菌能够侵染中蜂工蜂幼虫和雄蜂幼虫；通过small RNA-seq和生物信息学方法在意蜂幼虫肠道中鉴定到515个保守miRNA和45个新miRNA，揭示miR-4331-y和ame-miR-927b等DEmiRNA可能参与调控意蜂幼虫肠道响应球囊菌胁迫的应答；证实通过饲喂mimic（agomir）和inhibitor对意蜂幼虫进行miRNA的过表达和敲减具有充分的可行性，ame-miR-13b通过调控Egfr、P450和Dorsal等靶标影响意蜂幼虫对球囊菌胁迫的应答过程；首次在中蜂幼虫肠道中鉴定到537个miRNA，揭示miR-1277-x和miR-26-x等DEmiRNA可能通过调控Jak-STAT信号通路等参与介导中蜂幼虫肠道对球囊菌胁迫的应答；证实通过饲喂mimic和inhibitor对中蜂幼虫的miRNA进行过表达和敲减的方法可行；筛选和验证出中蜂幼虫应答球囊菌胁迫的2个关键DEmiRNA；通过比较转录组分析揭示免疫基因的数量差异是中蜂幼虫和意蜂幼虫的球囊菌抗性差异产生的重要原因之一，中蜂幼虫与球囊菌在长期的协同进化中可能通过提高能量利用限制病原增殖。本项目通过深入解析意蜂幼虫和中蜂幼虫肠道应答球囊菌胁迫的miRNA差异表达谱及DEmiRNA的调控作用，在分子水平揭示了两种蜜蜂幼虫的球囊菌抗性差异产生的机制，筛选和验证的关键DEmiRNA有望作为新型生物标志物和分子靶点应用于养蜂生产中白垩病的诊断和治疗。因此，本项目具有重要的科学意义和较好的应用潜力。