侵染中华蜜蜂6日龄幼虫的蜜蜂球囊菌的微小RNA差异表达谱及调控网络  

Differential expression pattern and regulation network of microRNAs in Ascosphaera apis invading Apis cerana cerana 6-day-old larvae

作　　者：熊翠玲 杜宇 冯睿蓉 蒋海宾 史小玉 王海朋 范小雪 王杰 祝智威 范元婵 陈华枝 周丁丁 郑燕珍 陈大福 郭睿 Cuiling Xiong;Yu Du;Ruirong Feng;Haibin Jiang;Xiaoyu Shi;Haipeng Wang;Xiaoxue Fan;Jie Wang;Zhiwei Zhu;Yuanchan Fan;Huazhi Chen;Dingding Zhou;Yanzhen Zheng;Dafu Chen;Rui Guo(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian Province,China)

机构地区：[1]福建农林大学动物科学学院(蜂学学院),福建福州350002

出　　处：《微生物学报》2020年第5期992-1009,共18页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31702190);现代农业产业技术体系建设专项资金(CARS-44-KXJ7);福建省教育厅中青年教师教育科研项目(JAT170158);福建农林大学杰出青年科研人才计划(xjq201814);福建农林大学科技创新专项基金(CXZX2017342,CXZX2017343);福建省大学生创新创业训练计划(201910389011,201810389029,201810389082)。

摘　　要：【目的】蜜蜂球囊菌(Ascosphaeraapis,简称球囊菌)是专性侵染蜜蜂幼虫的致死性真菌病原。MicroRNA(miRNA)作为一类重要的基因表达调控因子,能够广泛参与真菌及其宿主的相互作用过程。本研究通过比较分析球囊菌孢子(AaCK)和侵染中华蜜蜂(Apis cerana cerana,简称中蜂)6日龄幼虫肠道内的球囊菌(AaT)的smallRNA(sRNA)组学数据对球囊菌的差异表达miRNA(differentiallyexpressed miRNA,DEmiRNA)、靶mRNA及二者间的调控网络进行全面解析,旨在揭示miRNA介导的球囊菌对中蜂幼虫的侵染机制。【方法】对于球囊菌侵染的中蜂6日龄幼虫肠道的small RNA-seq(sRNA-seq)数据,利用BLAST工具连续比对东方蜜蜂(Apiscerana)和球囊菌的参考基因组筛滤得到AaT的sRNA组学数据。分别将AaCK和AaT的sRNA组学数据比对miRBase数据库,对球囊菌侵染宿主前后miRNA的数量和结构特征进行分析。联用RNAhybrid+svm_light、Miranda和TargetScan软件预测AaCK vs AaT比较组中DEmiRNA的靶mRNA,进而利用相关生物信息学软件对上述靶mRNA进行GO分类和KEGG代谢通路富集分析。通过Cytoscape软件对DEmiRNA-mRNA调控网络进行可视化。利用Stem-loop RT-PCR、RT-qPCR和分子克隆验证测序结果的可靠性。【结果】在AaCK和AaT中分别鉴定到380和387个miRNA。结构特征分析结果显示,AaCK和AaT的mi RNA皆集中分布在18–25 nt,且首位碱基主要偏向于U。AaCKvsAaT比较组共有270个DEmiRNA,包含155个上调miRNA和115个下调miRNA,分别靶向结合6091和6145个mRNA。GO分类结果显示,上述靶mRNA主要涉及代谢进程、细胞进程、应激反应等15个生物学进程;细胞、细胞组分、细胞器等12个细胞组分;催化活性、结合、转运子活性等11个分子功能。KEGG代谢通路富集分析结果显示,上述靶mRNA富集在123条代谢通路,参与对氨基酸代谢、碳水化合物代谢以及核苷酸代谢等物质代谢,氧化磷酸化、硫代谢、氮代谢等能量代谢,�[Objective]This study aimed to reveal miRNA-mediated mechanism underlying Ascosphaera apis infection of Apis cerana cerana larvae.[Methods]Small RNA(sRNA)dataset of A.apis during infection(AaT)was screened out from sRNA-seq data from Ascosphaera apis-infected A.c.cerana 6-day-old larval guts.The filtered sRNA datasets from the purified spores(AaCK)and AaT were aligned against miRBase using Blast,followed by analyses of number and structural characteristics of pathogen miRNAs before and after Ascosphaera apis infection.Prediction,GO categorization and KEGG pathway enrichment analysis of targets of DEmiRNAs were conducted using related software.The regulation network between DEmiRNAs and corresponding targets was visualized using Cytoscape.Stem-loop RT-PCR,qPCR and molecular cloning were used to verify the reliability of our sequencing data.[Results]Totally,380 and 387 miRNAs were identified in AaCK and AaT,respectively.The length of Ascosphaera apis miRNAs were mainly distributed between 18 nt and 25 nt;and the first base had a U bias.There were 155 up-regulated and 115 down-regulated miRNAs in AaCK vs AaT,targeting 6091 and 6145 mRNAs.Targets of DEmiRNAs were involved in 15 biological processes,12 cell components and 11 molecular functions.Additionally,these targets were engaged in 123 pathways,regulating material metabolisms,energy metabolisms and signaling pathways.Moreover,complex regulation networks existed between DEmiRNA and corresponding targets,among them miR-29-x,miR-250-x,miR-4968-y,miR-11200-x,novel-m0023-5p,novel-m0130-5p and novel-m0135-5p can target m RNAs associated with cysteine proteinase,DNA methyltransferases and chitinase;miR-7-x,miR-9-z,miR-319-y and miR-5951-y can simultaneously regulate MAPK signaling pathway;miR-250-x may be involved in cross-kingdom regulation between A.apis and A.c.cerana larvae.[Conclusion]Our results revealed DEmiRNAs may participate in the infection process of A.apis via regulating targets associated with material and energy metabolisms,pathogen proliferation,virulenc

关 键 词：蜜蜂球囊菌 中华蜜蜂 幼虫 微小RNA 调控网络 侵染机制 跨界调控 

分 类 号：S895[农业科学—特种经济动物饲养]