摘要
【目的】蜜蜂球囊菌(Ascosphaera apis,球囊菌)专性侵染蜜蜂幼虫而导致白垩病。本研究旨在通过small RNA-seq(sRNA-seq)技术和生物信息学方法对球囊菌纯化菌丝(AaM)和纯化孢子(AaS)进行深度测序和比较分析,明确球囊菌菌丝miRNA和孢子miRNA的数量、结构和表达谱差异,并揭示菌丝和孢子共有miRNA、特有miRNA和差异表达miRNA(differentially expressed miRNA,DEmiRNA)及其靶mRNA与球囊菌菌丝和孢子生长、发育和病原致病性的潜在关系。【方法】实验室条件下获得纯培养的球囊菌,利用sRNA-seq技术对AaM和AaS分别进行测序,通过对原始读段(raw reads)进行过滤和质控获得有效标签序列(clean tags)。通过Venn分析筛选菌丝和孢子共有miRNA和特有miRNA。根据P≤0.05且|log2 fold change|≥1的标准筛选AaM vs AaS的DEmiRNA。对上述共有miRNA、特有miRNA和DEmiRNA的靶mRNA进行预测,并对靶mRNA进行GO及KEGG数据库注释。根据靶向结合关系构建DEmiRNA和靶mRNA的调控网络。利用RT-qPCR验证测序数据的可靠性。【结果】AaM和AaS中分别得到12982320和12708832条raw reads,经过滤和质控分别得到10800101和9888848条clean tags。AaM中miRNA的长度介于18-26 nt,AaS中miRNA的长度介于18-24 nt,分布miRNA数量最多的长度均为18 nt,AaM和AaS中首位碱基为U的miRNA数量最多。AaM和AaS中表达量最高的miRNA均为miR6478-x、miR10516-x和miR482-x。菌丝和孢子共有miRNA靶向结合5946个mRNA,二者特有miRNA分别靶向结合6141和6346个mRNA。共有miRNA的靶mRNA主要参与代谢进程、细胞进程和催化活性等42个功能条目,以及翻译、碳水化合物代谢和能量代谢等120条通路。AaM vs AaS比较组包含93个DEmiRNA,可靶向结合6090个mRNA,这些靶mRNA可注释到38个功能条目和120条通路。DEmiRNA与靶mRNA之间形成较为复杂的调控网络,miR-4968-y位于调控网络的中心且能够靶向结合多达118个mRNA。RT-qPCR结果显示5个DEmiRNA的表达趋�
【Objective】Ascosphaera apis exclusively infects honeybee larvae,resulting in chalkbrood disease.The objective of this study is to clarify the differences of number,structure and expression pattern of miRNAs between A.apis mycelium and spore based on deep sequencing and comparative analysis of purified mycelia(AaM)and spores(AaS)using small RNA-seq(sRNA-seq)and bioinformatics,and reveal the potential relationship between common miRNAs,specific miRNAs,differentially expressed miRNAs(DEmiRNAs)and their target mRNAs and the growth and development of mycelium and spore as well as pathogenesis of A.apis.【Method】The pure culture of A.apis was gained under lab condition.AaM and AaS were respectively sequenced using sRNA-seq technology.Clean tags were obtained after filtration and quality control of raw reads.Common miRNAs and specific miRNAs in AaM and AaS were screened out using Venn analysis.DEmiRNAs in the AaM vs AaS comparison group were filtered out following the criteria of P≤0.05 and|log2 fold change|≥1.Target mRNAs of common miRNAs,specific miRNAs and DEmiRNAs were predicted using related bioinformatic software.Target mRNAs mentioned above were respectively annotated to GO database and KEGG database.The regulatory network of DEmiRNAs and target mRNAs was constructed on basis of target binding relationship,followed by visualization with Cytoscape.RT-qPCR was conducted to verify the reliability of the sequencing data.【Result】In total,12982320 and 12708832 raw reads were produced from AaM and AaS,and after strict quality control,10800101 and 9888848 clean tags were gained,respectively.The length of specific miRNAs in AaM was distributed among 18-26 nt,while that in AaS was distributed among 18-24 nt.Additionally,most of the miRNAs were distributed in 18 nt.MiRNAs with the first base U in both AaM and AaS were the most abundant.MiRNAs with the highest expression levels in both AaM and AaS were miR6478-x,miR10516-x and miR482-x.These common miRNAs could target 5946 mRNAs,while specific miRNAs in AaM an
作者
陈华枝
祝智威
蒋海宾
王杰
范元婵
范小雪
万洁琦
卢家轩
熊翠玲
郑燕珍
付中民
陈大福
郭睿
CHEN HuaZhi;ZHU ZhiWei;JIANG HaiBin;WANG Jie;FAN YuanChan;FAN XiaoXue;WAN JieQi;LU JiaXuan;XIONG CuiLing;ZHENG YanZhen;FU ZhongMin;CHEN DaFu;GUO Rui(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002)
出处
《中国农业科学》
CAS
CSCD
北大核心
2020年第17期3606-3619,共14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(CARS-44-KXJ7)
福建省自然科学基金(2018J05042)
福建省教育厅中青年教师教育科研项目(JAT170158)
福建农林大学杰出青年科研人才计划(xjq201814)
福建农林大学科技创新专项(CXZX2017342,CXZX2017343)
福建农林大学优秀硕士学位论文资助基金(陈华枝)。
