关键词: 意大利蜜蜂, 中肠, 发育, 差异表达基因, 竞争性内源RNA, 调控网络

Abstract:

【Objective】The whole transcriptome sequencing of Apis mellifera ligustica 7- and 10-day-old workers’ midguts (Am7 and Am10) was previously conducted. In this study, the differential expression profile and regulation network of genes were investigated to reveal the molecular mechanism underlying the midgut development.【Method】Gene expressions were calculated based on FPKM (fragments per kilobase of transcript per million mapped reads) algorithm, and differentially expressed genes (DEGs) were gained following the standard |log₂ fold change|≥1 and P≤0.05. Target mRNAs of ame-miR-6001-3p were predicted utilizing TargetFinder. Annotations of all DEGs in GO and KEGG databases were performed using related software. In addition, DEGs enriched in 13 signaling pathways including AMPK, P13K-Akt, Wnt, cAMP, FoxO, Hippo, mTOR, Jak-STAT, Toll-like receptor, TGF-beta, Notch, MAPK and NF-κB, as well as DEGs targeted by ame-miR-6001-3p were screened out, followed by visualization of enrichment networks and regulation networks with Cytoscape. Finally, Stem loop RT-PCR and RT-qPCR were used to verify the expression and differential expression pattern of ame-miR-6001-3p and DEGs in Am7 and Am10.【Result】A total of 1 038 DEGs were identified in Am7 vs Am10 comparison group, including 515 up- and 523 down-regulated genes, respectively. These DEGs were associated with cellular process, metabolic process and catalytic activity, and significantly enriched in some material and energy metabolisms such as oxidative phosphorylation, amino sugar and nucleotide sugar metabolisms, fatty acid metabolism and purine metabolism, indicative of the active cellular and metabolic activities. Expression cluster analysis suggested that 20, 18, 15 and 14 DEGs were respectively enriched in AMPK signaling pathway, PI3K-Akt signaling pathway, endocytosis and Hippo signaling pathway. In addition, 57 DEGs were enriched in the aforementioned 13 signaling pathways associated with growth and development as well as immune defense, among them one DEG was enriched in several signaling pathways. Moreover, regulation network analysis showed that 54 up-regulated genes and 44 down-regulated genes were targets of ame-miR-6001-3p, respectively; these up-regulated genes were enriched in 43 pathways including inositol phosphate metabolism, Hippo signaling pathway, glutathione metabolism and insulin signaling pathway, while these down-regulated genes were enriched in 20 pathways including Hippo signaling pathway, metabolic pathways, glutathione metabolism and arachidonic acid metabolism. Moreover, RT-qPCR result showed that the variation trend of six randomly selected DEGs were consistent with that in sequencing data, confirming the reliability of DEGs. Finally, ame-miR-6001-3p was definitely expressed and significantly down-regulated in Am10.【Conclusion】In this work, the expression pattern of DEGs and the regulation network between DEGs and ame-miR-6001-3p as well as the potential role of DEGs during the developmental process of A. m. ligustica worker’s midgut were deeply analyzed. The results revealed that the DEGs may participate in various signaling pathways including TGF-beta, Wnt, Hippo, Notch, PI3K-Akt, mTOR, AMPK, NF-κB signaling pathways, thus affecting the growth and development as well as immune defense of the midgut; DEGs were likely to regulate several signaling pathways such as insulin signaling pathway during the midgut development via formation regulation networks with significantly down-regulated ame-miR-6001-3p.

Key words: Apis mellifera ligustica, midgut, development, differentially expressed gene (DEG), competitive endogenous RNA, regulation network