关键词: 意大利蜜蜂, 竞争性内源RNA, 长链非编码 RNA, 时空表达谱, 蜜蜂球囊菌

Abstract: 【Aim】 The objective of this study is to provide a reference and basis for further exploring the regulatory function and mechanism of lncRNA15837 in Apis mellifera ligustica by analyzing the regulatory mode and role of lncRNA15837 and detecting its expression patterns in different developmental stages and different adult tissues, and in larvae of A. mellifera ligustica in response to Ascosphaera apis infection.【Methods】 Based on the obtained high-quality transcriptome data from the midguts of the 7- and 10-day-old adult workers of A. mellifera ligustica, Miranda, RNAhybrid and TargetScan software was employed to predict lncRNA15837-targeted miRNAs and miRNA-targeted mRNAs, and construction and visualization of the competing endogenous RNA (ceRNA) regulatory network were performed using Cytoscape v3.7.1 software. By using Blast tool, target mRNAs were aligned to GO and KEGG databases to gain their corresponding functional and pathway annotations, respectively. The expression of lncRNA15837 in different tissues (antennae, hypopharyngeal glands, brain, cuticle, midgut, fat body and venom gland) of the newly emerged adult workers was confirmed by RT-PCR. RT-qPCR was utilized to detect the expression levels of lncRNA15837 in different developmental stages of workers (eggs, 3-day-old larvae, 1- and 2-day-old prepupae, 4-day-old pupae, and 1-, 2-, 6-, 12-, 15- and 18-day-old adult workers), in different tissues (antennae, pharyngeal glands, brain, cuticle, midgut, fat body and venom gland) of the newly emerged adult workers, and in the guts of the 4-, 5- and 6-day-old larvae after infection of the 3-day-old larvae with Ascosphaera apis. 【Results】 LncRNA15837 could target 29 miRNAs such as ame-miR-21-x and ame-miR-0046-3p and further target 1 559 mRNAs, forming a complex ceRNA network. The aforementioned target mRNAs could be annotated to 436 GO terms, among them 114 target mRNAs were involved in 139 molecular function-related terms, 115 target mRNAs were involved in 257 biological process-related terms, 67 target mRNAs were involved in 40 cellular component-related terms, and could be annotated to 224 KEGG pathways. Among the 1 559 mRNAs, 28 target mRNAs were annotated to cellular immune pathways such as endocytosis, phagocytes and lysosomes, and 157 target mRNAs were annotated to humoral immune pathways such as JAK-STAT, Toll and Imd, NF-kappa B and Toll-like receptor signaling pathways. LncRNA15837 was differentially expressed in eggs, larvae, prepupae, pupae and adults of workers, and its expression level was the lowest in the 1-day-old prepupae but the highest in the 3-day-old larvae. LncRNA15837 was differentially expressed in the antennae, venom gland, brain, midgut, hypopharyngeal glands, fat body and cuticle of the newly emerged adult workers, with the lowest expression level in the fat body and the highest expression level in the venom gland. The expression level of lncRNA15837 in the A. apis-inoculated group was up-regulated in the 4-day-old larval gut and significantly up-regulated in the 5- and 6-day-old larval guts, as compared to that in the larval gut in uninoculated group.【Conclusion】 LncRNA15837 of A. mellifera ligustica potentially regulates cellular immune pathways such as endocytosis, phagocytes and lysosomes, and humoral immune pathways including JAK-STAT, Toll and Imd, NF-kappa B and Toll-like receptor signaling pathways through ceRNA network. lncRNA15837 is specifically and highly expressed in the 3-day-old worker larvae and venom gland of adult workers. The expression of lncRNA15837 in the larval gut of workers is activated by Ascosphaera apis infection, suggesting that lncRNA15837 is a putative regulator in larval response of A. mellifera ligustica to Ascosphaera apis infection.

Key words: Apis mellifera ligustica, competing endogenous RNAs, long non-coding RNA, spatiotemporal expression profile; Ascosphaera apis