中华蜜蜂幼虫肠道响应球囊菌胁迫的microRNA应答分析

MicroRNA responses in the larval gut of Apis cerana cerana to Ascosphaera apis stress

摘要:

[目的]蜜蜂球囊菌（Ascosphaera apis，简称球囊菌）是一种能够侵染中华蜜蜂（Apis cerana cerana，简称中蜂）幼虫的致死性真菌病原。微小RNA（microRNA，miRNA）可通过在转录后水平靶向抑制或降解mRNA而参与宿主与病原互作过程。本研究旨在对球囊菌胁迫的中蜂6日龄幼虫肠道的差异表达miRNA（DEmiRNA）及其靶基因进行深入分析，进而揭示DEmiRNA在中蜂响应球囊菌胁迫应答过程中的作用。[方法]利用Illumina MiSeq平台对正常及球囊菌胁迫的中蜂6日龄幼虫肠道（AcCK和AcT）进行测序，通过相关生物信息学软件预测DEmiRNA及其靶基因。通过Blast将靶基因注释到GO和KEGG数据库。利用Cytoscape软件构建DEmiRNA与其靶mRNA的调控网络。通过Stem-loop RT-PCR和qPCR验证测序数据的可靠性。[结果]本研究共预测出537个miRNA，其长度分布介于16-35 nt之间，且不同长度的miRNA首位碱基偏向性差异明显。通过Stem-loop RT-PCR证实了10个novel miRNA的表达。AcCK vs AcT比较组共有54个DEmiRNA，包含31个上调和23个下调miRNA，可分别靶向结合6170和8199个靶基因。GO分类结果显示上调和下调miRNA的靶基因分别涉及47和47个条目，富集基因数最多的皆为结合细胞进程和催化活性。KEGG代谢通路（pathway）富集分析结果表明上调和下调miRNA的靶基因分别富集在134和126条pathway，富集基因数最多的均为内吞作用和内质网中的蛋白质加工。调控网络分析结果表明，DEmiRNA及其靶mRNA形成十分复杂的调控关系；31个DEmiRNA可靶向结合51个与泛素介导的蛋白水解相关的mRNA，18个DEmiRNA可靶向结合14个与Jak-STAT信号通路相关的mRNA；miR-1277-x、miR-26-x、miR-27-y、miR-30-x、miR-6052-x等16个miRNA共同参与了上述两条免疫通路的调控。最后，随机挑选3个DEmiRNA进行qPCR验证，结果证明了测序数据的可靠性。[结论]本研究提供了中蜂幼虫肠道在球囊菌胁迫后期的miRNA的表达谱和差异表达信息，揭示了球囊菌与宿主之间在miRNA组学水平存在复杂的互作。miR-6052-x和miR-1277-x作为调控网络的核心可能通过影响细胞凋亡参与宿主的免疫防御，miR-26-x和miR-30-x可能通过调控Jak-STAT信号通路参与宿主的胁迫应答。本研究筛选出的关键DEmiRNA有望作为治疗白垩病的分子靶标。

关键词:中华蜜蜂;幼虫;球囊菌;microRNA;靶基因;调控网络

Abstract:

[Objective] Ascosphaera apis is a lethal fungal pathogen for Apis cerana cerana larvae. microRNA can participate in host-pathogen interaction processes by inhibition or degradation of mRNA via targeting at post-transcriptional level. The aim of this study was to analyze the differentially expressed miRNAs (DEmiRNAs) and their target genes in the 6-day-old larval gut of A. c. cerana under A. apis stress and reveal DEmiRNAs' roles in the stress response process. [Methods] Normal and A. apis-challenged 6-day-old larval guts of A. c. cerana (AcCK and AcT) were sequenced using Illumina MiSeq platform, followed by prediction and analysis of DEmiRNAs and their target genes using related softwares. Target genes of DEmiRNAs were annotated to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases with Blast. Regulation networks between DEmiRNAs and target mRNAs were constructed using Cytoscape. Stem-loop RT-PCR and qPCR were conducted to verify the reliability of sequencing data. [Results] Deep sequencing of larval gut samples generated 537 miRNAs, the length of which was distributed between 16 nt and 35 nt. The first base bias of miRNAs with various length had apparent difference. The expression of 10 novel miRNAs were validated using Stem-loop RT-PCR. There were 54 DEmiRNAs in AcCK vs. AcT comparison group, including 31 up-regulated and 23 down-regulated miRNAs, which can respectively link 6170 and 8199 target genes. GO classification suggested that target genes of up-and down-regulated miRNAs were respectively involved in 47 and 47 terms, and the largest ones were binding, cellular process, and catalytic activity. KEGG pathway enrichment analysis demonstrated that target genes of up-and down-regulated miRNAs were respectively engaged in 134 and 126 pathways, and the mostly enriched ones were endocytosis and protein processing in endoplasmic reticulum. Analysis of regulation networks revealed that very complex regulation relationships existed between DEmiRNAs and corresponding target mRNAs; 31 miRNAs could bind 51 mRNAs associated with ubiquitin mediated proteolysis, and 18 miRNAs can bind 14 Jak-STAT signaling pathway-associated mRNAs; a total of 16 miRNAs, such as miR-1277-x, miR-26-x, miR-27-y, miR-30-x and miR-6052-x, can participate in regulating both of the above-mentioned immune pathways. Finally, three DEmiRNAs were randomly selected for qPCR, the result verified the reliability of our transcriptome sequencing data. [Conclusion] We first provided the expression profile and differential expression information of A. c. cerana miRNAs during the late stage of A. apis stress, revealed the complex interactions between A. apis and host at transcriptome level. As the core of regulation networks, miR-6052-x and miR-1277-x were likely to participate in host immune defense by affecting apoptosis, while miR-26-x and miR-30-x may join host responses to A. apis stress via regulation of Jak-STAT signaling pathway. Key DEmiRNAs screened in our study are expected to be used as potential molecular targets for chalkbrood control.

Key words:Apis cerana cerana;larvae;Ascosphaera apis;microRNA;target gene;regulation network