微小RNA介导东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂的分子机制

The Mechanism Underlying MicroRNAs-Mediated Nosema ceranae Infection to Apis mellifera ligustica Worker

【目的】通过对东方蜜蜂微孢子虫(Nosema ceranae)纯化孢子与侵染意大利蜜蜂(Apis mellifera ligustica,简称意蜂)工蜂的东方蜜蜂微孢子虫的差异表达miRNA(DEmiRNA)及其靶mRNA进行系统分析,筛选、分析和探讨病原毒力因子和侵染因子相关的DEmiRNA及调控网络,在miRNA组学层面揭示东方蜜蜂微孢子虫对意蜂的侵染机制。【方法】利用small RNA-seq(sRNA-seq)技术对东方蜜蜂微孢子虫感染7 d和10 d的意蜂工蜂中肠和东方蜜蜂微孢子虫纯化孢子(NcCK)进行深度测序,通过连续比对rRNA数据库、西方蜜蜂(Apis mellifera)基因组和东方蜜蜂微孢子虫基因组筛滤出处于侵染过程的东方蜜蜂微孢子虫(NcT1和NcT2)数据和东方蜜蜂微孢子虫孢子的测序数据。根据P≤0.05,|log2 fold change|≥1的标准,通过比较分析筛选出各比较组中的差异表达miRNA(differentially expressed miRNA,DEmiRNA)。通过相关生物信息学软件对DEmiRNA进行表达谱分析,靶mRNA预测及功能和代谢通路注释,以及调控网络的构建与分析。通过Stem-loop RT-qPCR验证DEmiRNA的差异表达趋势及测序数据的可靠性。【结果】NcCK vs NcT1、NcCK vs NcT2和NcT1 vs NcT2比较组分别包含164、122和60个DEmiRNA。Venn分析结果显示,3个比较组共有的上调和下调miRNA分别为5和6个。上述DEmiRNA分别预测出1885、1733和1524个靶mRNA。这些靶mRNA分别注释到27、25和26个功能条目,其中注释数量最多的是新陈代谢进程、催化活性、细胞进程、结合和细胞。上述靶mRNA可分别注释到84、84和84条代谢通路,其中注释数量最多的是代谢途径、核糖体和次级代谢产物生物合成。此外,对于NcCK vs NcT1、NcCK vs NcT2和NcT1 vs NcT2中的DEmiRNA,分别有35、26和12个靶向结合MAPK信号通路相关靶mRNA,分别有49、40和17个DEmiRNA靶向结合糖酵解/糖异生通路相关靶mRNA。进一步分析发现,东方蜜蜂微孢子虫的DEmiRNA参与调控蓖麻毒素B凝集素、细胞凋亡抑制因子、极管蛋白和孢壁蛋白等病原毒力因子的基因表达,以及己糖激酶、ATP/ADP移位酶、ABC转运蛋白和转录因子ste12等侵染因子的基因表达。【结论】通过对东方蜜蜂微孢子虫纯化孢子与侵染意蜂工蜂的东方蜜蜂微孢子虫进行深入细致的miRNA组学分析和探讨,解析了病原侵染过程的miRNA差异表达谱,揭示了东方蜜蜂微孢子虫可能通过调节相应miRNA的表达水平对蓖麻毒素B凝集素、细胞凋亡抑制因子、极管蛋白和孢壁蛋白等毒力因子及己糖激酶、ATP/ADP移位酶、ABC转运蛋白和转录因子ste12等侵染因子基因表达进行调控,从而适应宿主细胞内的环境并促进自身的增殖与侵染。

【Objective】Differentially expressed miRNAs(DEmiRNAs)and their target mRNAs in purified spores of Nosema ceranae and N.ceranae infecting Apis mellifera ligustica worker were systematically analyzed,followed by screening,investigation and exploration of DEmiRNAs and corresponding regulatory networks associated with pathogenic virulence factors and infection factors to reveal the mechanism of N.ceranae infection to A.m.ligustica worker.【Method】A.m.ligustica workers’midguts at 7 d and 10 d post infection(dpi)with N.ceranae and purified spores of N.ceranae(NcCK)were deeply sequenced using small RNA-seq(sRNA-seq),followed by screening out datasets of N.ceranae during the infection process(NcT1 and NcT2)and purified spores through sequential mapping to rRNA database,Apis mellifera genome and N.ceranae genome.Based on the criteria of P≤0.05,|log2 fold change|≥1,DEmiRNAs in each comparison group were filtered out by comparative analysis.Expression profiling of DEmiRNAs,prediction of target mRNAs,function and pathway annotations of target mRNAs,and construction and survey of regulatory networks were performed by relevant bioinformatic software.The differential expression trend of DEmiRNAs and the reliability of sequencing data were verified by RT-qPCR.【Result】There were 164,122 and 60 DEmiRNAs in NcCK vs NcT1,NcCK vs NcT2 and NcT1 vs NcT2 comparison groups.Venn analysis showed that 5 up-regulated miRNAs and 6 down-regulated ones were shared by these three comparison groups.In total,1885,1733 and 1524 target mRNAs of DEmiRNAs were respectively predicted.These targets were annotated to 27,25,and 26 functional terms,respectively,with the most abundant annotations being metabolic processes,catalytic activity,cellular processes,binding and cell.These targets were as well respectively annotated to 84,84,and 84 pathways,among them the largest groups were metabolic pathway,ribosome,and secondary metabolite biosynthesis.In addition,35,26 and 12 target mRNAs associated with MAPK signaling pathway were targeted by DEmiRNAs in NcCK vs NcT1,NcCK vs NcT2 and NcT1 vs NcT2,while 49,40 and 17 target mRNAs related to glycolysis/gluconeogenesis pathway were targeted by DEmiRNAs in the aforementioned three comparison groups.Further analysis revealed that DEmiRNAs of N.ceranae were involved in regulating the expression of genes encoding virulence factors such as ricin B lectin,apoptosis inhibitor,polar tube protein and spore wall protein;and genes encoding infection factors such as hexokinase and ATP/ADP transferase,ABC transporter and transcription factor ste12.【Conclusion】The differential expression profile of miRNAs in N.ceranae infecting A.m.ligustica worker was parsed via in-depth and detailed investigation and exploration.N.ceranae may regulate the expression level of genes encoding virulence factors including ricin B lectin,apoptosis inhibitor,polar tube protein and spore wall protein,as well as genes encoding infection factors such as hexokinase,ATP/ADP transferase,ABC transporter and transcription factor ste12 corresponding miRNAs,by controlling the expression of corresponding miRNAs,thus adapting to the environment within the host cell and promoting its proliferation and infection.