侵染中华蜜蜂6日龄幼虫的蜜蜂球囊菌的微小RNA差异表达谱及调控网络

【目的】蜜蜂球囊菌(Ascosphaeraapis,简称球囊菌)是专性侵染蜜蜂幼虫的致死性真菌病原。MicroRNA(miRNA)作为一类重要的基因表达调控因子,能够广泛参与真菌及其宿主的相互作用过程。本研究通过比较分析球囊菌孢子(AaCK)和侵染中华蜜蜂(Apis cerana cerana,简称中蜂) 6日龄幼虫肠道内的球囊菌(AaT)的smallRNA(sRNA)组学数据对球囊菌的差异表达miRNA(differentiallyexpressed miRNA,DEmiRNA)、靶mRNA及二者间的调控网络进行全面解析,旨在揭示miRNA介导的球囊菌对中蜂幼虫的侵染机制。【方法】对于球囊菌侵染的中蜂6日龄幼虫肠道的small RNA-seq (sRNA-seq)数据,利用BLAST工具连续比对东方蜜蜂(Apiscerana)和球囊菌的参考基因组筛滤得到AaT的sRNA组学数据。分别将AaCK和AaT的sRNA组学数据比对miRBase数据库,对球囊菌侵染宿主前后miRNA的数量和结构特征进行分析。联用RNAhybrid+svm_light、Miranda和Tar...

Differential expression pattern and regulation network of microRNAs in Ascosphaera apis invading Apis cerana cerana 6-day-old larvae

[Objective] This study aimed to reveal miRNA-mediated mechanism underlying Ascosphaera apis infection of Apis cerana cerana larvae. [Methods] Small RNA (sRNA) dataset of A. apis during infection (AaT) was screened out from sRNA-seq data from Ascosphaera apis-infected A. c. cerana 6-day-old larval guts. The filtered sRNA datasets from the purified spores (AaCK) and AaT were aligned against miRBase using Blast, followed by analyses of number and structural characteristics of pathogen miRNAs before and after Ascosphaera apis infection. Prediction, GO categorization and KEGG pathway enrichment analysis of targets of DEmiRNAs were conducted using related software. The regulation network between DEmiRNAs and corresponding targets was visualized using Cytoscape. Stem-loop RT-PCR, qPCR and molecular cloning were used to verify the reliability of our sequencing data. [Results] Totally, 380 and 387 miRNAs were identified in AaCK and AaT, respectively. The length of Ascosphaera apis miRNAs were mainly distributed between 18 nt and 25 nt; and the first base had a U bias. There were 155 up-regulated and 115 down-regulated miRNAs in AaCK vs AaT, targeting 6091 and 6145 mRNAs. Targets of DEmiRNAs were involved in 15 biological processes, 12 cell components and 11 molecular functions. Additionally, these targets were engaged in 123 pathways, regulating material metabolisms, energy metabolisms and signaling pathways. Moreover, complex regulation networks existed between DEmiRNA and corresponding targets, among them miR-29-x, miR-250-x, miR-4968-y, miR-11200-x, novel-m0023-5p, novel-m0130-5p and novel-m0135-5p can target mRNAs associated with cysteine proteinase, DNA methyltransferases and chitinase; miR-7-x, miR-9-z, miR-319-y and miR-5951-y can simultaneously regulate MAPK signaling pathway; miR-250-x may be involved in cross-kingdom regulation between A. apis and A. c. cerana larvae. [Conclusion] Our results revealed DEmiRNAs may participate in the infection process of A. apis via regulating targets associated with material and energy metabolisms, pathogen proliferation, virulence, and several signaling pathways; several key miRNAs including miR-7-x were potential targets for chalkbrood control.