中华蜜蜂6日龄幼虫响应蜜蜂球囊菌侵染的长链非编码RNA应答研究

Long non-coding RNA response of 6-day-old Apis cerana cerana larvae to Ascosphaera apis infection

摘要:

[目的] 本研究旨在探究长链非编码RNA（long non-coding RNA，lncRNA）在中华蜜蜂（Apis cerana cerana，简称中蜂）6日龄幼虫应答蜜蜂球囊菌（Ascosphaera apis，简称球囊菌）侵染过程中的差异表达谱及调控作用。[方法] 利用链特异性cDNA建库的RNA-seq技术对未被侵染及球囊菌侵染的中蜂6日龄幼虫肠道（AcCK和AcT）进行深度测序。通过相关生物信息学软件分析lncRNA的结构特征和表达谱。筛选并分析差异表达lncRNA（differentially expressed lncRNA，DElncRNA）的顺式（cis）作用及竞争性内源RNA（competing endogenous RNA，ceRNA）调控网络。采用RT-qPCR验证测序数据及DElncRNA差异变化趋势的可靠性。[结果] AcCK和AcT共预测出642个已知lncRNA和487个新lncRNA。与蛋白编码基因相比，上述中蜂lncRNA外显子数更少、长度更短且表达量更低。43个antisense lncRNA与40个正义链mRNA之间互补配对。AcCK和AcT比较组包含367个上调lncRNA和268个下调lncRNA。有194个DElncRNA潜在调控461个上下游基因，并涉及细胞进程、代谢进程和催化活性等38个功能条目以及氨基酸代谢、内吞作用和MAPK等191条通路。此外，有180个DElncRNA可靶向结合50个DEmiRNA，进而调控6365个mRNA；三者之间形成较为复杂的ceRNA调控网络。[结论] 中蜂6日龄幼虫肠道的部分lncRNA可作为antisense lncRNA参与应答球囊菌侵染；部分DElncRNA可通过cis作用调节物质代谢和免疫途径相关的上下游基因，从而介导宿主的侵染应答；TCONS_00010661和TCONS_00003104等DElncRNA可通过ceRNA网络调控Jak-STAT和氧化磷酸化等通路及富集基因，进而参与宿主的侵染应答。

关键词:中华蜜蜂;幼虫;蜜蜂球囊菌;长链非编码RNA;竞争性内源RNA;免疫应答

Abstract:

[Objective] This study aims to investigate the differential expression pattern of long non-coding RNAs (lncRNAs) and their regulatory function involved in Apis cerana cerana 6-day-old larval gut response to Ascosphaera apis infection. [Methods] Un-infected and Ascosphaera apis-infected 6-day-old larval guts of Apis cerana cerana (AcCK and AcT) were sequenced using strand-specific cDNA library-based RNA-seq technology. Structural characteristics and expression pattern of lncRNAs were analyzed using related bioinformatic softwares. DElncRNAs were screened followed by investigation of their cis-acting role and competitive endogenous RNA (ceRNA) network. RT-qPCR was conducted to verify the sequencing data and expression pattern of DElncRNAs. [Results] Here, 642 known lncRNAs and 487 novel lncRNAs were identified. Compared with mRNAs, these Apis cerana cerana lncRNAs were shorter in exon and intron length, fewer in exon number and lower in expression level. Additionally, 43 antisense lncRNAs were discovered to have complementary relationship with 40 sense-strand mRNAs. In AcCK vs. AcT comparison group, 367 up-regulated lncRNAs and 268 down-regulated ones were identified. In total, 194 DElncRNAs were found to potentially regulate 461 upstream and downstream genes, which were annotated to 38 functional terms such as cellular process, metabolic process and catalytic activity, as well as 191 pathways including amino acid metabolism, endocytosis and MAPK signaling pathways. Moreover, 180 DElncRNAs can target 50 DEmiRNAs, further regulating 6365 mRNAs; additionally, complex regulatory networks existed among them. [Conclusion] These results demonstrated that partial antisense lncRNAs may participate in host response to Ascosphaera apis infection; some DElncRNAs may regulate upstream and downstream genes relative to material metabolism and immune-associated pathways, thus mediating host Ascosphaera apis-response; a portion of DElncRNAs including TCONS_00010661 and TCONS_00003104 were likely to regulate Jak-STAT signaling pathway and oxidative phosphorylation and corresponding enriched genes via ceRNA networks, further participating in the response of host to Ascosphaera apis invasion.

Key words:Apis cerana cerana;larva;Ascosphaera apis;long non-coding RNA;competitive endogenous RNA;immune response