关键词: 意大利蜜蜂, 中肠, 环状RNA, 调控网络, 发育

Abstract:

【Objective】Circular RNA (circRNA) plays a primary role in alternative splicing, transcription regulation and expression regulation of parental gene. The objective of this study is to investigate the profile expression of circRNAs and differentially expressed circRNAs (DEcircRNAs) during the developmental process of the midguts of Apis mellifera ligustica workers, and to explore the role of DEcircRNAs in the development of midgut at the transcriptional level. 【Method】 On basis of the whole transcriptome data from 7- and 10-day-old worker’s midguts of A. m. ligustica (Am7 and Am10), find_circ software was used to predict circRNAs based on the filtered sequencing data. The circRNA expression level was normalized by RPM algorithm. Differential expression analysis for circRNAs was conducted via DEGseq software following standards fold change≥2.0, P<0.05 and false discovery rate (FDR) <0.05. Source genes of DEcircRNAs were annotated to GO and KEGG databases to gain function and pathway annotations by using BLAST. DEcircRNAs-miRNAs and DEcircRNAs-miRNAs-mRNAs networks were predicted with TargetFinder software and visualized using Cytoscape v.3.2.1 software. RT-qPCR was conducted to verify the reliability of sequencing data.【Result】 On average, 19 616 356 anchors reads were obtained from each A. m. ligustica worker’s midgut sample. Pearson correlations between different biological repeats within Am7 and Am10 groups were ≥0.950. In total, 256 DEcircRNAs including 105 up-regulated circRNAs and 151 down-regulated circRNAs were predicted. Novel_circ_009675 and novel_circ_013879 were highly expressed in Am7 and Am10, respectively. Source genes of DEcircRNAs could be annotated to 32 GO terms including binding, single-organism process and cellular process, among them 35, 35 and 7 source genes were involved in catalytic activity, metabolic process and stress response. Additionally, these source genes could be annotated to 35 KEGG pathways, in which 5, 5 and 4 source genes were associated with Hippo signaling pathway, endocytosis and phagosome, respectively; further investigation showed that 1, 2 and 2 source genes could be annotated to material metabolisms such as phosphoinositol metabolism, starch and sucrose metabolism and galactose metabolism; 5, 4, 3, 1 and 1 source genes could be annotated to immune signaling pathways including endocytosis, phagosome, lysosome, ubiquitin-mediated proteolysis and MAPK signaling pathway, respectively. These results suggested that the corresponding DEcircRNA was involved in the development, metabolism and immune defense of the midgut of A. m. ligustica. DEcircRNA-miRNA regulation network analysis showed that 141 DEcircRNAs could link to 107 miRNAs, most of these DEcircRNAs could only bind to 1-2 miRNAs, but novel_circ_011577 and novel_circ_010719 could respectively bind to 32 and 28 miRNAs. In addition, the number of DEcircRNAs combined with mir-136-y, ame-miR-6001-3p and mir-136-y was the highest (15, 14 and 14, respectively), which indicated that the corresponding DEcircRNA could play roles during the developmental process of A. m. ligustica worker’s midgut as competing endogenous RNAs. Furthermore, DEcircRNAs-ame-miR-6001-3p-mRNA network was constructed and analyzed, and the result indicated that 14 DEcircRNAs could jointly link to ame-miR-6001-3p, implying they were likely to indirectly regulate division and differentiation of stem cells in A. m. ligustica worker’s midgut via regulation of ame-miR-6001-3p. Six DEcircRNAs were randomly selected for RT-qPCR assay, the result showed the alteration trend of expression levels of 5 DEcircRNAs was consistent with that of the sequencing data, which proved the reliability of trancriptome data.【Conclusion】Through the deep investigation of DEcircRNAs during the developmental process of A. m. ligustica worker’s midgut, the expression profile and differential expression information of circRNAs in the development of midgut of worker bee were provided, and the role of DEcircRNAs in the development of midgut was revealed. It provides a basis for the screening and functional study of key circRNAs associated with the development of the midgut.

Key words: Apis mellifera ligustica, midgut, circRNAs, regulation network, development