蜜蜂球囊菌菌丝和孢子中环状RNA的鉴定及比较分析  

Identification and comparison of circular RNAs in Ascosphaera apis mycelium and spore

作　　者：陈华枝 蒋海宾 祝智威 范元婵 许雅静 孙明会 刘佳美 熊翠玲 郑燕珍 付中民 徐国钧[1,2] 陈大福 郭睿 Huazhi Chen;Haibin Jiang;Zhiwei Zhu;Yuanchan Fan;Yajing Xu;Minghui Sun;Jiamei Liu;Cuiling Xiong;Yanzhen Zheng;Zhongmin Fu;Guojun Xu;Dafu Chen;Rui Guo(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian Province,China;Apitherapy Research Institution,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian Province,China;Engineering Research Center of Processing and Application of Bee Products of Ministry of Education,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 [2]福建农林大学蜂疗研究所,福建福州350002 [3]福建农林大学蜂产品加工与应用教育部工程研究中心,福建福州350002

出　　处：《微生物学报》2021年第5期1299-1314,共16页Acta Microbiologica Sinica

基　　金：国家现代农业产业技术体系建设专项资金(CARS-44-KXJ7);福建农林大学硕士生导师团队项目(郭睿);福建农林大学科技创新专项(CXZX2017342,CXZX2017343);福建省病原真菌与真菌毒素重点实验室(福建农林大学)开放课题;福建农林大学优秀硕士学位论文资助基金(陈华枝)。

摘　　要：【目的】本研究旨在明确蜜蜂球囊菌(Ascosphaera apis,简称球囊菌)菌丝和孢子中环状RNA(circular RNA,circRNA)的数量、种类和表达谱差异,并探讨共有circRNA、特有circRNA和差异表达circRNA(differentially expressed circRNA,DEcircRNA)在菌丝与孢子中的潜在作用。【方法】基于前期获得的球囊菌菌丝(AaM)和孢子(AaS)的高质量RNA-seq数据,利用find_circ软件预测circRNA。通过Venn分析筛选AaM和AaS的共有circRNA和特有circRNA。根据P≤0.05且|log2 fold change|≥1的标准筛选AaM vs.AaS比较组的DEcircRNA。通过比对GO和KEGG数据库对circRNA的来源基因进行功能和通路注释。利用TargetFinder软件预测circRNA靶向结合的miRNA及miRNA靶向结合的mRNA。采用Cytoscape软件对竞争性内源RNA(competing endogenous RNA,ceRNA)调控网络进行可视化。通过RT-qPCR对DEcircRNA进行验证。【结果】AaM和AaS中分别含有13210156和19011000条短序列读段(anchors reads),其中分别有6124922和11392886条能够比对上球囊菌参考基因组。在AaM和AaS中分别鉴定到1868个和2225个circRNA,二者共有的circRNA为1098个,AaM的特有circRNA为770个,AaS的特有circRNA为1127个。AaM和AaS的circRNA长度主要介于1000–2000 nt,基因间区circRNA为主要环化类型。AaM vs.AaS比较组包含456个上调circRNA和97个下调circRNA。共有circRNA的来源基因可注释到29个功能条目和14类通路;AaM的特有circRNA的来源基因可注释到31个功能和17类通路;AaS的特有circRNA的来源基因可注释到34个功能条目和16类通路;DEcircRNA的来源基因可注释到29个功能条目和40条通路。调控网络分析结果显示,36个共有circRNA靶向4个miRNA进而调控6个与内吞作用通路相关的靶mRNA;4(255)个AaM(AaS)的特有circRNA靶向2(2)个miRNA进而调控8(2)个次级代谢产物生物合成通路相关的靶mRNA;9个DEcircRNA靶向2个DEmiRNA进而调控3个MAPK信号通路相关的DEmRNA。RT-qPCR结果显示10个DEcircRNA的表达趋[Objective]The objective of this study is to clarify the differences of number,type and expression profile of circRNAs between Ascosphaera apis mycelium and spore,and discuss the potential role of common circRNAs,specific circRNAs and differentially expressed circRNAs(DEcircRNAs).[Methods]Based on previously gained high-quality RNA-seq data of A.apis mycelium(AaM)and spore(AaS),circRNAs were predicted using find_circ software.Common circRNAs and specific circRNAs were filtered via Venn analysis.DEcircRNAs of AaM vs AaS were screened following the standard of P≤0.05 and|log2 fold change|≥1.Function and pathway annotation of source genes of circRNAs was done via alignment against GO and KEGG databases.CircRNA-targeted miRNAs and miRNA-targeted mRNAs were predicted using TargetFinder software.Cytoscape software was used to visualize competing endogenous RNA(ceRNA)regulation network.DEcircRNAs were verified by RT-qPCR.[Results]There were 13210156 and 19011000 anchors reads in AaM and AaS,respectively,among them 6124922 and 11392886 ones can be mapped to the reference genome of A.apis.In AaM and AaS groups,1868 and 2225 circRNA were respectively identified,and the numbers of common circRNAs,specific circRNAs in AaM and specific circRNAs in AaS were 1098,770 and 1127,respectively.In addition,the length of circRNAs in AaM and AaS was mainly distributed among 1000–2000 nt,and the most abundant circularization type was intergenic circRNA.Further,456 upregulated circRNAs and 97 downregulated circRNAs were contained in AaM vs AaS comparison group.Source genes of common circRNAs were annotated to 29 functional terms and 14 pathway classifications;source genes of specific circRNAs in AaM were annotated to 31 functional terms and 17 pathway classifications;source genes of specific circRNAs in AaS were annotated to 34 functional terms and 16 pathway classifications;source genes of DEcircRNAs were annotated to 29 functional terms and 40 pathways.ceRNA regulation network analysis showed that 36 common circRNAs can target fo

关 键 词：白垩病 蜜蜂球囊菌 菌丝 孢子 环状RNA 微小RNA 竞争性内源RNA 

分 类 号：S895.137[农业科学—特种经济动物饲养]