关键词: 意大利蜜蜂, 中肠, 发育, 长链非编码RNA, 上下游基因

Abstract:

【Objective】Long non-coding RNA (lncRNA) plays an important role in regulation of gene expression, epigenetics and cell cycle in eukaryotes. The objective of this study is to investigate the expression profile and role of lncRNAs in the developmental process of Apis mellifera ligustica worker’s midgut. 【Method】In this study, 7- and 10-day-old worker’s midguts of A. m. ligustica (Am7, Am10) were sequenced using RNA-seq technology and strand-specific library construction method. Using Perl script, raw reads were filtered to obtain clean reads with high-quality. Bowtie tool was used to compare clean reads to the ribosome database, and TopHat2 software was employed to compare unmapped clean reads to the reference genome. CPC and CNCI softwares were utilized to predict coding capacity of the transcripts. RT-PCR was performed to identify partial lncRNAs. Investigation of differentially expressed lncRNAs (DElncRNAs) was carried out with edgeR, followed by prediction of upstream and downstream genes, for which GO and KEGG pathway enrichment analyses were performed. RNAhybrid, Miranda and TargetScan softwares were utilized together to predict target miRNAs of DElncRNAs and target genes of miRNAs, and DElncRNAs-miRNAs-mRNAs regulation networks were visualized via Cytoscape. Finally, RT-qPCR was conducted to verify reliability of the sequencing data.【Result】134 802 058 and 147 051 470 raw reads were gained from deep sequencing of Am7 and Am10, respectively, and after stringent filtration, 134 166 157 and 146 293 288 were obtained. In total, 6 353 lncRNAs were predicted, and 3 890 DElncRNAs were obtained based on expression calculation, including 2 005 up-regulated lncRNAs and 1 885 down-regulated lncRNAs. The result of RT-PCR suggested the expected signal bands could be amplified from 8 lncRNAs, implying their true existence. There were 1 793 upstream and downstream genes of DElncRNAs, which were involved in 42 GO terms, including metabolic processes, developmental processes, cellular processes, stress responses, immune system processes and so forth. They were also associated with 251 KEGG pathways, including material metabolism pathways such as carbon metabolism, purine metabolism and fatty acid biosynthesis; energy metabolism pathways such as sulfur metabolism, methane metabolism and oxidative phosphorylation; signaling pathways such as Hippo, Wnt and Notch signaling pathways; cellular immune pathways such as lysosome, endocytosis and ubiquitin mediated proteolysis; humoral immune pathways such as MAPK, Jak-STAT and NF-kappa B pathways, these results demonstrated the DElncRNAs were involved in the material and energy metabolism, cell life activity and immunity regulation in the developmental process of A. m. ligustica worker’s midgut. Further analysis showed TCONS_00020918 might play a regulatory part in the nutrient absorption and caste differentiation in the worker’s midgut. Analysis of regulation networks demonstrated that complex networks existed between DElncRNAs and target miRNAs and mRNAs, partial DElncRNAs lie in the central of the networks and link many miRNAs, and partial miRNAs could be bound by many DElncRNAs, which indicated that these DElncRNAs might play an important role during the developmental process of the worker’s midgut. Finally, 5 DElncRNAs were randomly selected for RT-qPCR assay, and the result proved the reliability of sequencing data in this study.【Conclusion】DElncRNA is widely involved in the metabolism, cellular activity and immune regulation of A. m. ligustica worker’s midgut, and plays a role as a competitive endogenous RNA (ceRNA). The results provide the necessary data support for the screening and functional study of key lncRNA.

Key words: Apis mellifera ligustica, midgut, development, long non-coding RNA, upstream and downstream genes