中华蜜蜂幼虫应答蜜蜂球囊菌侵染中 ace-miR-2478-y及其靶基因的表达模式和潜在作用

Expression profiles and putative roles of ace-miR-2478-y and its target gene in the response of Apis cerana cerana larvae to Ascosphara apis infection

摘要:

背景 蜜蜂球囊菌( Ascosphaera apis)是一种专性寄生真菌病原体，其感染专门针对蜜蜂幼虫，并通常可导致宿主罹患白垩病而死亡。目前，有关蜜蜂球囊菌在侵染过程中的miRNA的表达模式及其靶基因的潜在作用极为有限。 目的 检测中华蜜蜂( Apis cerana cerana)幼虫肠道中ace-miR-2478-y及其靶基因类固醇激素受体(steroid hormone receptor) ERR 1在蜜蜂球囊菌( Ascosphaera apis)侵染过程中的表达模式，为解析ace-miR-2478-y应答蜜蜂球囊菌侵染的分子调控机制提供依据。 方法 通过stem-loop RT-PCR和Sanger测序验证蜜蜂球囊菌接种前后工蜂幼虫肠道中ace-miR-2478-y的真实表达及序列准确性。利用生物信息学软件预测ace-miR-2478-y的靶mRNA，采用Cytoscape软件构建ace-miR-2478-y与其靶mRNA的调控网络。利用BLAST工具对靶mRNA序列进行同源性比对以鉴定其对应的靶基因，并基于GO与KEGG数据库获取这些基因的功能注释及通路信息。通过双荧光素酶报告基因实验验证ace-miR-2478-y与其靶基因 ERR 1的结合关系。利用RT-qPCR检测蜜蜂球囊菌接种的幼虫肠道中ace-miR-2478-y及其靶基因 ERR 1的相对表达量。 结果 stem-loop RT-PCR结果显示ace-miR-2478-y在蜜蜂球囊菌接种前后的中华蜜蜂幼虫肠道均扩增出目的片段，Sanger测序结果显示片段序列与前期测序结果完全一致。ace-miR-2478-y共靶向233条mRNA，对应110个靶基因，靶基因可注释到生物调控、细胞和结合等23条GO功能条目，以及Wnt信号通路、蛋白质吸收与消化等125条KEGG通路。双荧光素酶报告基因结果显示ace-miR-2478-y与其靶基因 ERR 1之间存在真实结合关系。RT-qPCR结果显示，相较于未侵染组，ace-miR-2478-y在蜜蜂球囊菌侵染后的1 dpi表达量显著上升，在2 dpi和3 dpi的表达量显著下降。靶基因 ERR 1的表达趋势与ace-miR-2478-y相反。相较于未侵染组， ERR 1在1 dpi的表达量显著下调，在2 dpi和3 dpi的表达量显著上调。 结论 ace-miR-2478-y通过负调控靶基因 ERR 1的表达响应蜜蜂球囊菌的侵染。研究结果为中华蜜蜂幼虫响应蜜蜂球囊菌侵染的机制研究提供参考。

Abstract:

Background Ascosphaera apis is an obligate parasitic fungal pathogen whose infectivity displays a marked specificity for honeybee larvae, typically resulting in lethal chalkbrood disease. However, the miRNA expression profiles of A. apis during host infection and the functions of their target genes remain largely unexplored. Objective We investigate the expression patterns of ace-miR-2478-y and its target gene steroid hormone receptor ( ERR 1) in the larval gut of Apis cerana cerana during Ascosphaera apis infection, aiming to lay a foundation for elucidating the molecular regulatory mechanisms of ace-miR-2478-y in response to A. apis infection. Methods stem-loop RT-PCR and Sanger sequencing were employed to verify the true expression of ace-miR-2478-y and the accuracy of its sequence in the larval gut before and after inoculation with A. apis. The target mRNAs of ace-miR-2478-y were predicted by bioinformatics software, and the regulatory network of ace-miR-2478-y and its target mRNAs was constructed in Cytoscape. The target mRNA sequences were subjected to homology alignment using the BLAST tool to identify their corresponding genes. Functional annotations and pathway information for these genes were then obtained based on the GO and KEGG databases. The binding relationship between ace-miR-2478-y and ERR 1 was verified by the dual luciferase reporter gene assay. RT-qPCR was employed to determine the relative expression of ace-miR-2478-y and ERR 1 in the guts of larvae inoculated with A. apis. Results The results of stem-loop RT-PCR showed that the target fragment of ace-miR-2478-y was amplified from the guts of A. c. cerana larvae before and after inoculation with A. apis. Sanger sequencing results showed that the fragment sequence was completely consistent with the sequencing results. Ace-mi-2478-y targeted 233 mRNAs, corresponding to 110 target genes. These target genes could be annotated to 23 GO terms such as biological regulation, cell, and binding and 125 KEGG pathways such as the Wnt signaling pathway and protein absorption and digestion. The results of dual luciferase reporter gene assay showed a real binding relationship between ace-miR-2478-y and its target gene ERR 1. RT-qPCR results showed that compared with that in the uninfected group, the expression of ace-miR-2478-y increased significantly at one day post inoculation (dpi) of A. apis and decreased significantly at two dpi and three dpi. The expression of the target gene ERR 1 showed an opposite trend, being significantly down-regulated at 1 dpi and significantly up-regulated at 2 dpi and 3 dpi. Conclusion Ace-miR-2478-y responded to the infection of A. apis by negatively regulating the expression of the target gene ERR 1. The results provide a reference for deciphering the mechanism by which A. c. cerana larvae respond to the A. apis infection.