胁迫意大利蜜蜂幼虫肠道的球囊菌的转录组分析

摘要

[目的]本研究旨在通过趋势分析对胁迫意大利蜜蜂Apis mellifera ligustica(简称意蜂)幼虫肠道的球囊菌Ascosphaera apis的差异表达基因(DEGs)进行转录组分析.[方法]将纯化的球囊菌孢子配制为1×10 7孢子/mL的饲料饲喂意蜂3日龄幼虫,利用Illumina HiSeq 2500平台对球囊菌胁迫的意蜂幼虫肠道cDNA进行测序,将过滤得到的有效读段(clean reads)映射(mapping)到核糖体数据库及意蜂参考基因组,最后将未映射上的reads映射到本课题组组装并注释的球囊菌参考转录组.利用STEM软件对DEGs进行趋势分析.利用WEGO软件对显著表达模式中的DEGs进行GO富集分析.利用Blastall对显著表达模式中的DEGs进行KEGG代谢通路富集分析.最后,通过对随机选取的6个DEGs进行RT-qPCR分析,对RNA-seq数据进行验证.[结果]球囊菌胁迫意蜂幼虫肠道样品的Illumina测序共得到球囊菌的25 454 076条原始读段(raw reads),经过滤得到24 909 820条clean reads,Q30均在93.46％以上.趋势分析结果显示,19 893个DEGs聚类为8个表达模式,其中有12 151个DEGs聚类为3个表现为显著上调趋势的表达模式.GO富集分析结果显示,表现上调趋势的DEGs富集在40个GO term,富集基因数最多的为细胞进程(cellular process)(2 601 unigenes),其次为代谢进程(metabolic process)(2 553 unigenes)和细胞(cell)(2 522 unigenes).KEGG代谢通路富集分析结果显示,上调趋势中的DEGs富集在1 19个代谢通路上,其中富集基因数最多的是核糖体(ribosome)(213 unigenes),其次为氨基酸生物合成(biosynthesis of amino acids)(154 unigenes)和内质网蛋白加工(protein processing in endoplasmic reticulum)(130unigenes).共有48个DEGs富集在MAPK信号通路上,聚类分析结果显示,这些DEGs随着胁迫时间的延长表达水平逐渐增强.RT-qPCR结果显示,6个DEGs的表达水平变化趋势与RNA-seq 数据一致,证明了本研究中的转录组数据真实可靠.[结论]本研究为在分子水平揭示球囊菌的致病机理提供了重要信息,也为阐释球囊菌胁迫意蜂幼虫过程中的病原-宿主互作机制奠定了基础.%[Aim] This research is designed to conduct transcriptomic analysis of the differentially expressed genes (DEGs) of Ascosphaera apis stressing larval guts of Apis mellifera ligustica via trend analysis.[Methods] The purified A.apis spores at a concentration of 1 × 107 spores/mL was used to feed 3-day-old larvae of A.m.ligustica,and then the cDNA of stressed larval guts was sequenced at Illumina HiSeq 2500 platform.After filtration,the clean reads were used to mapping the ribosome database and the reference genome of A.m.ligustica,and the unmapped reads were used to mapping the reference transcriptome of A.apis assembled previously.The STEM software was used to analyze the gene expression patterns.Gene ontology (GO) enrichment analysis for DEGs involved in significant expression patterns was performed using WEGO software.KEGG enrichment analysis for DEGs associated with significant expression patterns was carried out by using Blastall.Finally,RT-qPCR analysis of six randomly selected DEGs was performed to validate the RNA-seq data.[Results] The RNA-seq of A.apis produced 25 454 076 raw reads,and after filtration,24 909 820 clean reads with Q30 above 93.46％ were obtained.Trend analysis results showed that 19 893 DEGs were grouped into eight gene expression patterns,among which 12 151 DEGs were assigned to three expression patterns with significantly upregulated expression trend.GO enrichment analysis results indicated that all DEGs within siguificantly up-regulated expression patterns were enriched in 40 GO terms,and the mostly enriched one was cellular process (2 601 unigenes),followed by metabolic process (2 553 unigenes) and cell (2 522 unigenes).KEGG enrichment analysis result displayed that all DEGs within significantly up-regulated expression patterns were enriched in 119 metabolism pathways,and the mostly enriched one was ribosome (213 unigenes),followed by biosynthesis of amino acids (154 unigenes) and protein processing in endoplasmic reticulum (130 unigenes).Furthermore,it was found that 48 DEGs were enriched in MAPK signaling pathway,and the cluster result suggested that the expression levels of these DEGs increased as the stressing time prolonged.RT-qPCR results demonstrated that the expression patterns of the six DEGs were consistent with those of RNA-seq data,confirming that our transcriptome data are credible.[Conclusion] The findings in this study not only provide the key information for uncovering the pathogenesis of A.apis at the molecular level,but also lay a foundation for clarifying the pathogen-host interaction in A.m.ligustica under the stress of A.apis.