Data_Sheet_2_Transcriptional dynamics and regulatory function of milRNAs in Ascosphaera apis invading Apis mellifera larvae.PDF

In the present study, small RNA (sRNA) data from Ascosphaera apis were filtered from sRNA-seq datasets from the gut tissues of A. apis-infected Apis mellifera ligustica worker larvae, which were combined with the previously gained sRNA-seq data from A. apis spores to screen differentially expressed milRNAs (DEmilRNAs), followed by trend analysis and investigation of the DEmilRNAs in relation to significant trends. Additionally, the interactions between the DEmilRNAs and their target mRNAs were verified using a dual-luciferase reporter assay. In total, 974 A. apis milRNAs were identified. The first base of these milRNAs was biased toward U. The expression of six milRNAs was confirmed by stem–loop RT-PCR, and the sequences of milR-3245-y and milR-10285-y were validated using Sanger sequencing. These miRNAs grouped into four significant trends, with the target mRNAs of DEmilRNAs involving 42 GO terms and 120 KEGG pathways, such as the fungal-type cell wall and biosynthesis of secondary metabolites. Further investigation demonstrated that 299 DEmilRNAs (novel-m0011-3p, milR-10048-y, bantam-y, etc.) potentially targeted nine genes encoding secondary metabolite-associated enzymes, while 258 (milR-25-y, milR-14-y, milR-932-x, etc.) and 419 (milR-4561-y, milR-10125-y, let-7-x, etc.) DEmilRNAs putatively targeted virulence factor-encoded genes and nine genes involved in the MAPK signaling pathway, respectively. Additionally, the interaction between ADM-B and milR-6882-x, as well as between PKIA and milR-7009-x were verified. Together, these results not only offer a basis for clarifying the mechanisms underlying DEmilRNA-regulated pathogenesis of A. apis and a novel insight into the interaction between A. apis and honey bee larvae, but also provide candidate DEmilRNA–gene axis for further investigation.

本研究从感染蜜蜂球囊菌（Ascosphaera apis）的意大利蜜蜂（Apis mellifera ligustica）工蜂幼虫肠道组织的小RNA测序（sRNA-seq）数据中，筛选得到源自蜜蜂球囊菌的小RNA（sRNA）数据，并将其与此前获取的蜜蜂球囊菌孢子的sRNA-seq数据进行整合，以筛选差异表达milRNAs（DEmilRNAs）；随后对DEmilRNAs开展趋势分析，并探究其与显著表达趋势的关联。此外，采用双荧光素酶报告基因实验验证了DEmilRNAs与其靶mRNA之间的互作关系。本研究共鉴定得到974个蜜蜂球囊菌milRNAs，此类milRNAs的首个碱基偏好性为尿嘧啶（U）。通过茎环反转录聚合酶链反应（stem–loop RT-PCR）验证了6个milRNAs的表达水平，并利用桑格测序（Sanger sequencing）确认了milR-3245-y与milR-10285-y的序列信息。这些milRNAs可分为4个显著表达趋势，其靶mRNA涉及42个基因本体（GO）术语及120条京都基因与基因组百科全书（KEGG）通路，例如真菌型细胞壁与次级代谢产物生物合成等通路。进一步分析显示，299个DEmilRNAs（如novel-m0011-3p、milR-10048-y、bantam-y等）潜在靶向9个编码次级代谢相关酶的基因；258个DEmilRNAs（如milR-25-y、milR-14-y、milR-932-x等）及419个DEmilRNAs（如milR-4561-y、milR-10125-y、let-7-x等）分别推定靶向毒力因子编码基因及9个参与丝裂原活化蛋白激酶（MAPK）信号通路的基因。此外，本研究验证了ADM-B与milR-6882-x、PKIA与milR-7009-x之间的互作关系。综上，本研究结果不仅为阐明蜜蜂球囊菌DEmilRNAs调控的致病机制提供了理论依据，也为解析蜜蜂球囊菌与蜜蜂幼虫之间的互作关系提供了新视角，同时为后续研究提供了候选DEmilRNA-基因调控轴。