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

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AaMvs.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。AaMvs.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的表达趋势与测序数据一致，证实了测序数据的可靠性。[结论] 球囊菌菌丝和孢子的共有circRNA、特有circRNA和DEcircRNA可能通过调控来源基因表达和充当ceRNA的方式调节球囊菌的物质和能量代谢、内吞作用、次级代谢产物生物合成和MAPK信号通路，进而影响球囊菌菌丝生长、孢子萌发和致病性。

Abstract:

[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 four miRNAs, further regulating six mRNAs related to endocytosis. Additionally, four (255) specific circRNAs in AaM (AaS) can target two (two) miRNAs, further regulating eight (two) mRNAs related to biosynthesis of secondary metabolites; nine DEcircRNAs can target two DEmiRNAs, further regulating three DEmRNAs associated with MAPK signal pathway. The result of RT-qPCR displayed that the expression trend of 10 DEcircRNAs was consistent with that in the sequencing result, verifying the reliability of our sequencing data. [Conclusion] Common circRNAs, specific circRNAs and DEcircRNAs in A. apis mycelium and spore may regulate material and energy metabolisms, endocytosis, biosynthesis of secondary metabolites, and MAPK signaling pathway through controlling the expression of source genes and serving as ceRNAs, further affecting the mycelium growth, spore germination, and pathogenicity of A. apis.