关键词: 东方蜜蜂微孢子虫, 意大利蜜蜂, 微小RNA, 侵染机制, 调控网络

Abstract:

【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, 1 885, 1 733 and 1 524 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.

Key words: Nosema ceranae, Apis mellifera ligustica, microRNA (miRNA), infection mechanism, regulatory network