意大利蜜蜂幼虫肠道在球囊菌侵染前期的差异表达microRNA及其调控网络  

Differentially Expressed MicroRNAs and Their Regulation Networks in Apis mellifera ligustica Larval Gut During the Early Stage of Ascosphaera apis Infection

作　　者：郭睿[1] 杜宇[1] 童新宇 熊翠玲[1] 郑燕珍[1] 徐国钧[1] 王海朋 耿四海 周丁丁 郭意龙 吴素珍 陈大福[1] GUO Rui;DU Yu;TONG XinYu;XIONG CuiLing;ZHENG YanZhen;XU GuoJun;WANG HaiPeng;GENG SiHai;ZHOU DingDing;GUO YiLong;WU SuZhen;CHEN DaFu(College of Bee Science,Fujian Agriculture and Forestry University,Fuzhou 350002)

机构地区：[1]福建农林大学蜂学学院,福州350002

出　　处：《中国农业科学》2019年第1期166-180,共15页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31702190);国家现代农业产业技术体系建设专项资金(CARS-44-KXJ7);福建省科技计划(2018J05042);福建省教育厅中青年教师教育科研项目(JAT170158);福建农林大学科技创新专项基金(CXZX2017343);福建省大学生创新创业训练计划(201710389058;201810389082)

摘　　要：【目的】微小RNA(microRNA,miRNA)是一类重要的基因表达调控因子,可影响宿主与病原间的互作过程。蜜蜂球囊菌(Ascosphaera apis)是一种特异性侵染蜜蜂幼虫的致死性真菌病原。本研究旨在对意大利蜜蜂(Apis mellifera ligustica,简称意蜂)幼虫肠道在球囊菌胁迫前期的差异表达miRNA(differentially expressed miRNA,DEmiRNA)及其靶基因进行深入分析,在miRNA组学水平探究意蜂幼虫在球囊菌侵染前期的胁迫应答,并通过构建显著DEmiRNA的调控网络筛选出与宿主应答相关的关键miRNA。【方法】利用small RNA-seq(sRNA-seq)技术对正常及球囊菌侵染的意蜂4日龄幼虫肠道(AmCK和AmT)进行高通量测序,首先对原始数据进行质控和评估,随后将过滤后的数据与西方蜜蜂(Apis mellifera)参考基因组进行比对;将比对上的序列标签(tags)注释到miRBase数据库,得出已知miRNA的表达量;通过TPM(tags per million)算法对各样本中miRNA的表达量进行归一化处理,以|log_2 fold change|≥1且P≤0.05作为标准筛选得到显著DEmiRNA;利用TargetFinder软件预测显著DEmiRNA的靶基因,并对其进行GO和KEGG代谢通路(pathway)富集分析。利用Cytoscape软件对miRNA-mRNA调控网络进行可视化。最后,利用茎环反转录PCR(Stem-loop RT-PCR)和荧光定量PCR(qPCR)验证测序数据的可靠性。【结果】AmCK和AmT样品的测序分别得到13 553 302和10 777 534条原始读段(raw reads),经严格过滤后得到的有效读段(clean reads)数分别为13 186 921和10 480 913条。各样品的生物学重复间的Pearson相关性系数分别在0.9822和0.9508以上。共有10个显著DEmiRNA,包括4个上调miRNA和6个下调miRNA。显著DEmiRNA在AmT的整体表达水平低于AmCK。10个显著DEmiRNA可靶向结合3 788个靶基因,其中上调miRNA的1 240个靶基因可注释到GO数据库中的39个GO条目,主要富集在结合、细胞进程、代谢进程和应激反应等;下调miRNA的749个靶基因可注释到34个GO条目【Objective】Micro RNA(mi RNA) is a kind of key gene expression regulator, which can affect the interactions between host and pathogen. Ascosphaera apis is a lethal fungal pathogen that specifically infects honeybee larvae. The objective of this study is to analyze the differentially expressed miRNAs(DEmiRNAs) and their target genes in the Apis mellifera ligustica larval gut during the early infection stage of A. apis, reveal DEmiRNA’ roles in the stress responses of host at the miRNA omics level, and to screen the key miRNAs related to host response by constructing regulation networks of significant DEmiRNAs. 【Method】Normal and A. apis-infected 4-day-old larval gut of A. m. ligustica(Am CK and Am T) were deep-sequenced using small RNA-seq(sRNA-seq) technology, followed by quality-control of raw data and then mapping of the filtered data with the reference genome of Apis mellifera. The mapped tags were compared to the miRBase database to identify the expression of known mi RNAs. The expression of miRNAs in each sample was normalized by TPM(tags per million) algorithm and significant DEmiRNAs were gained according to the standard |log2 fold change|≥1 and P≤0.05. Target genes of significant DEmiRNAs were predicted utilizing TargetFinder, and then annotated to the GO and KEGG databases. Cytoscape was used to visualize the regulation networks between significant DEmiRNAs and target m RNAs. Finally, Stem-loop RT-PCR and qPCR were conducted to verify the reliability of the sequencing data.【Result】sRNA-seq of AmCK and AmT produced 13 553 302 and 10 777 534 raw reads, and after strict filtration, 13 186 921 and 10 480 913 clean reads were obtained, respectively. The Pearson correlation coefficients among different biological replicates in each sample were above 0.9822 and 0.9508. There were 10 significant DEmiRNAs including 4 up-regulated miRNAs and 6 down-regulated miRNAs, and the overall expression level of DEmiRNAs in AmT was lower than that in AmCK. In total, 10 significant DEmiRNAs could link 3 788 t

关 键 词：意大利蜜蜂 幼虫肠道 发育 差异表达微小RNA 调控网络 

分 类 号：S895[农业科学—特种经济动物饲养]