意大利蜜蜂幼虫肠道在球囊菌胁迫后期的差异表达微小RNA及其靶基因分析  

Comprehensive analysis of differentially expressed microRNAs and their target genes in the larval gut of Apis mellifera ligustica during the late stage of Ascosphaera apis stress

作　　者：郭睿[1] 杜宇[1] 周倪红 刘思亚 熊翠玲[1] 郑燕珍[1] 付中民[1] 徐国钧[1] 王海朋 耿四海 周丁丁 陈大福[1] GUO Rui;DU Yu;ZHOU Ni-Hong;LIU Si-Ya;XIONG Cui-Ling;ZHENG Yan-Zhen;FU Zhong-Min;XU Guo-Jun;WANG Hai-Peng;GENG Si-Hai;ZHOU Ding-Ding;CHEN Da-Fu(College of Bee Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建农林大学蜂学学院,福州350002

出　　处：《昆虫学报》2019年第1期49-60,共12页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31702190);现代农业产业技术体系建设专项资金(CARS-44-KXJ7);福建省科技计划项目(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;CXZX2017343)

摘　　要：【目的】蜜蜂球囊菌Ascosphaera apis(简称球囊菌)是一种特异性侵染蜜蜂幼虫肠道的致死性真菌病原。微小RNA(microRNA,mi RNA)是一种在转录后水平对m RNA进行负调控的关键调控因子。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后期的差异表达mi RNAs(DEmiRNAs)及其靶基因进行深入分析,为揭示DEmiRNAs在胁迫应答中的作用提供重要信息。【方法】利用small RNA-seq(s RNA-seq)技术对正常及球囊菌侵染的意蜂6日龄幼虫肠道(分别表示为Am CK和Am T)进行测序,通过相关生物信息学软件对DEmiRNAs进行预测和分析。利用TargetFinder软件预测DEmiRNAs的靶基因,然后利用Blast软件对靶基因进行GO和KEGG数据库的功能注释。通过Cytoscape软件构建DEmiRNAs与其靶m RNAs的调控网络。通过茎环实时荧光定量PCR(stem-loop RT-qPCR)验证测序数据的可靠性。【结果】Am CK和Am T的测序分别得到9 230 496和10 823 667条有效序列标签; Am CK vs Am T比较组中包含15个上调和6个下调mi RNAs,分别结合3 503和3 252个靶基因,它们可分别注释到40和39个GO terms以及104和99条代谢通路;进一步分析发现调控网络中的17个DEmiRNAs靶向结合116个与丝氨酸蛋白酶相关的m RNAs,14个DEmiRNAs靶向结合54个与泛素介导的蛋白水解相关的m RNAs。Stem-loop RT-qPCR验证结果显示随机选择的4个DEmiRNAs (mi R-251-x,mi R-9277-y,mi R-1672-x和mi R-4968-y)的表达量变化趋势与测序结果一致,表明测序数据真实可信。【结论】本研究率先对意蜂幼虫肠道在球囊菌胁迫后期的DEmiRNAs及其靶基因进行预测与分析,提供了mi RNAs的表达谱和差异表达信息。ame-miR-927b,mi R-429-y和mi R-8440-y等DEmiRNAs可能参与对丝氨酸蛋白酶的调控和对泛素介导的蛋白水解的调控,DEmiRNAs与靶基因之间存在复杂的调控网络关系。DEmiRNAs可参与到意蜂幼虫肠道与球囊菌间的互作。【Aim】Ascosphaera apis is a deadly fungal pathogen that specially infects the larval gut of honeybees.MicroRNA(miRNA)plays a key negative role in regulation of mRNA at the post-transcriptional level.This study aims to comprehensively analyze the differentially expressed miRNAs(DEmiRNAs)and their target genes in the larval gut of Apis mellifera ligustica during the late stage of A.apis stress,so as to provide important information for revealing the roles of DEmiRNAs in the stress response process.【Methods】Normal and A.apis challenged 6-day-old larval gut of A.m.ligustica(represented as AmCK and AmT,respectively)were sequenced using small RNA-seq(sRNA-seq)technology,followed by prediction and analysis of DEmiRNAs.Target genes of DEmiRNAs were predicted with TargetFinder,and annotations of target genes in GO and KEGG databases were performed using Blast.The regulation networks between DEmiRNAs and the target mRNAs were constructed using Cytoscape.The reliability of the sequencing data was verified by stem-loop RT-qPCR.【Results】Deep sequencing of AmCK and AmT samples respectively generated 9 230 496 and 10 823 667 clean tags.There were 15 up-regulated and 6 down-regulated miRNAs in AmCK vs AmT comparison group,binding 3 503 and 3 252 target genes,which could be annotated to 40 and 39 GO terms as well as 104 and 99 metabolic pathways,respectively.Further investigation indicated that 17 DEmiRNAs within the regulation networks could bind to 116 serine protease-related mRNAs,and 14 DEmiRNAs were capable of binding to 54 ubiquitin-mediated proteolysis associated mRNAs.The result of stem-loop RT-qPCR showed that the variation trends in the expression levels of randomly selected four DEmiRNAs(miR-251-x,miR-9277-y,miR-1672-x and miR-4968-y)were consistent with those in sRNA-seq data,indicating the credibility and reliability of our sequencing data.【Conclusion】Our study takes the lead in the prediction and investigation of DEmiRNAs and corresponding target genes in the larval gut of A.m.ligustica during the lat

关 键 词：意大利蜜蜂 蜜蜂球囊菌 胁迫应答 微小RNA 免疫防御 靶基因 

分 类 号：S895.1[农业科学—特种经济动物饲养]