LncRNA13164通过ace-miR-4968-y调节中华蜜蜂幼虫对蜜蜂球囊菌侵染的免疫应答

LncRNA13164 regulates immune response of Apis cerana cerana larvae to Ascosphaera apis infection via ace-miR-4968-y

摘要:

【目的】长链非编码RNA （long non-coding RNA，lncRNA）可通过竞争性内源RNA （competing endogenous RNA，ceRNA）等多种方式发挥重要的调控作用，但蜜蜂lncRNA的功能研究至今依然缺失，lncRNA在蜜蜂免疫应答中的作用尚不清楚。本研究旨在揭示中华蜜蜂（Apis cerana cerana，简称中蜂）幼虫响应蜜蜂球囊菌（Ascosphaera apis）侵染的免疫应答中lncRNA的调控功能及作用机制。笔者团队前期通过深度测序和生物信息学分析发现，lncRNA13164与ace-miR-4968存在靶向关系且潜在参与在中蜂幼虫对蜜蜂球囊菌侵染的应答。【方法】利用实时荧光定量PCR （real-time fluorescence quantitative polymerase chain reaction，RT-qPCR）检测蜜蜂球囊菌接种后中蜂幼虫肠道内lncRNA13164的表达谱。采用ILoc-LncRNA软件预测lncRNA13164的亚细胞定位。联用RNAhybrid、Miranda和TargetScan软件预测lncRNA13164靶向的miRNA及miRNA靶向的mRNA。通过PCR和RT-qPCR验证lncRNA13164和ace-miR-4968的真实表达及二者间的结合关系。通过饲喂dsRNA对蜜蜂球囊菌侵染的中蜂幼虫肠道内lncRNA13164进行RNAi，进而检测lncRNA13164的沉默效果及ace-miR-4968和3个免疫基因（stk、e3ul和or1）的相对表达量。【结果】相较于未接种组，lncRNA13164的表达量在蜜蜂球囊菌接种组4日龄幼虫肠道内上调，在5和6日龄幼虫肠道内显著上调。LncRNA13164可靶向ace-miR-4968等15个miRNA并形成1个调控网络。ace-miR-4968共靶向79个基因，可注释到17个基因本体论（gene ontology，GO）条目和85条京都基因和基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路。LncRNA13164与ace-miR-4968在中蜂幼虫肠道内均真实表达。相较于未接种组，接种组5日龄幼虫肠道内ace-miR-4968-y极显著下调表达，与lncRNA13164的表达趋势相反。相较于dsRNA-egfp饲喂组，dsRNA-lncRNA13164饲喂组5和6日龄幼虫肠道内lncRNA13164的表达量均极显著下调（P<0.01），沉默效率分别为66.05%和56.45%。沉默lncRNA13164后，ace-miR-4968的表达量在5日龄幼虫肠道内极显著上调，stk、e3ul和or1的表达量均显著下调（P<0.05）。【结论】饲喂dsRNA可有效沉默中蜂幼虫肠道内lncRNA13164的表达，lncRNA13164通过ace-miR-4968调控丝氨酸苏氨酸蛋白激酶（serine/threonine-protein kinase Doa isoform X4）基因stk、E3泛素蛋白连接酶（E3 ubiquitin-protein ligase，MYLIP）基因e3ul和抗氧化蛋白1亚型X6（oxidation resistance protein 1 isoform X6）基因or1表达，进而介导宿主对蜜蜂球囊菌侵染的免疫应答。

Abstract:

[Objective] Long non-coding RNA (lncRNA) usually functions as competing endogenous RNA (ceRNA) to play crucial regulatory roles. As no study of the function of bee lncRNA is available, the role of lncRNA in the immune response of bee is unclear. This study aims to reveal the regulatory function and mechanism of lncRNA in immune response of Apis ceranacerana larvae to Ascosphaera apis infection. Through deep sequencing and bioinformatics analysis, we have found that lncRNA targets ace-miR-4968 and involves in the response of A. c. cerana larvae to A. apis infection. [Methods] Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was performed to quantify the expression of lncRNA13164 in larval guts of A. c. cerana after A. apis inoculation. ILoc-LncRNA was employed to predict subcellular localization of lncRNA13164. RNAhybrid, Miranda, and TargetScan were applied to predict target miRNAs of lncRNA13164 and miRNA-targeted mRNAs. PCR and RT-qPCR were performed to validate expression of lncRNA13164 and ace-miR-4968 as well as their potential binding relationship. The larvae which had been infected with A. apis were fed with dsRNA for RNAi of lncRNA13164 in larval guts, followed by determination of the silencing effect on lncRNA13164 and relative expression of ace-miR-4968 and three immune genes (stk, e3ul, and or1). [Results] The expression of lncRNA13164 was up-regulated in guts of 4-day-old larvae and significantly up-regulated in guts of 5- and 6-day-old larvae in the inoculation group as compared with that in the non-inoculation group. LncRNA13164 targeted 15 miRNAs including ace-miR-4968, which formed a regulatory network. ace-miR-4968 putatively targeted 79 genes which were involved in 17 gene ontology (GO) terms and 85 Kyoto encyclopedia of genes and genomes (KEGG) pathways. Both lncRNA13164 and ace-miR-4968 were expressed in A. c. cerana larval gut. The expression of lncRNA13164 in guts of both 5- and 6-day-old larvae was significantly down-regulated as compared with that in the dsRNA-egfp-fed group, and silencing efficiencies were 66.05% and 56.45%, respectively. After the silencing of lncRNA13164, the expression of ace-miR-4968 was up-regulated (P<0.01) in guts of 5-day-old larvae, while expression of stk, e3ul, and or1 was down-regulated (P<0.05). [Conclusion] lncRNA13164 in A. c. cerana larval guts can be silenced through the feeding of specific dsRNA. lncRNA13164 regulates the expression of serine/threonine-protein kinase Doa isoform X4 gene stk, E3 ubiquitin-protein ligase (MYLIP) gene e3ul, and oxidation resistance protein 1 isoform X6 gnen or1 via ace-miR-4968 and further mediates the immune response of host to A. apis invasion.