关键词: 蜜蜂球囊菌, 长链非编码RNA, 顺式作用, 反义lncRNA, 竞争性内源RNA

Abstract:

【Objective】Long non-coding RNAs (lncRNAs), a kind of RNAs with highly conserved secondary and tertiary structures and a length of more than 200 nt, have no protein-coding potential. LncRNAs are widely involved in transcriptional and post-transcriptional regulations, such as dosage compensation, cell differentiation and growing development. The study aimed to perform analyses of cis regulation, antisense lncRNA regulation and competing endogenous RNA (ceRNA) regulation as well as related discussion based on previously obtained high-quality lncRNA omics dataset from mixed samples of Ascosphaera apis mycelia and spores, and further reveal the putative regulatory role of lncRNAs in A. apis.【Method】Protein-coding genes located at 100 kb upstream and downstream of A. apis lncRNAs were predicted on basis of the position of lncRNA genes; these upstream and downstream genes were annotated to GO and KEGG databases using Blast to gain function and pathway annotations. Target mRNAs of lncRNAs were identified using LncTar software and then annotated to KEGG and eggNOG databases. Target miRNAs of lncRNAs and target mRNAs of miRNAs were predicted using TargetFinder, followed by visualization of regulatory networks using Cytoscape v3.7.1. The expressions of partial lncRNAs, target miRNAs and target mRNAs within regulatory networks were validated using RT-PCR.【Result】A total of 5 852 upstream and downstream genes of A. apis lncRNAs were predicted. These upstream and downstream genes could be annotated to 48 functional terms such as cellular process, metabolic process and stress response, as well as 121 pathways including metabolic pathways, biosynthesis of secondary metabolites and biosynthesis of antibiotics, which indicated that part of A. apis lncRNAs could regulate the expression levels of upstream and downstream genes via cis-acting function, thus participating in regulation of basic cell activities including material and energy metabolisms. Seven lncRNAs were found to have sequence complementary pairing relationship with seven target mRNAs. Among them, five targets were annotated to hypothetical proteins in eggNOG database, while gene3444 could be annotated to nuclear pore complex protein An-Nup120 and hypothetical protein in KEGG database, suggesting that the aforementioned one antisense lncRNA might be involved in regulating biological processes including biosynthesis of nuclear pore complex protein. Additionally, 227 lncRNAs were predicted to have target binding relationship with 73 miRNAs; majority of these lncRNAs (79.02%) could only link to 1-2 miRNAs and partial miRNAs could be targeted by several lncRNAs. Furthermore, lncRNA-miRNA-mRNA regulatory networks associated with oxidative phosphorylation pathway and MAPK signaling pathway were constructed and analyzed, the results showed that 222 lncRNAs related to oxidative phosphorylation pathway could target 78 miRNAs and 50 mRNAs; as for MAPK signaling pathway, 222 associated lncRNAs could target 76 miRNAs and 46 mRNAs; the results suggested that part of A. apis lncRNAs were likely to regulate these two pathways via ceRNA mechanism, hence affecting energy synthesis, environmental adaptation as well as growth and development.【Conclusion】Partial lncRNAs may regulate a series of biological processes such as growth and development, material and energy mechanisms as well as environmental adaptation of A. apis; MSTRG.5393.1 as an antisense lncRNA may regulate biosynthesis of nuclear pore complex protein in A. apis.

Key words: Ascosphaera apis, long non-coding RNA (lncRNA), cis regulation, antisense lncRNA, competing endogenous RNA (ceRNA)