关键词: 意大利蜜蜂, 球囊菌, 幼虫肠道, RNAseq, 转录组, 差异表达基因

Abstract: 【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 1×10⁷spores/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 WEGOsoftware. 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 up-regulated expression trend. GO enrichment analysis results indicated that all DEGs within significantly 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.

Key words: Apis mellifera ligustica； Ascosphaera apis； larval gut, RNA-seq； transcriptome； differentially expressed genes (DEGs)