基于small RNA组学分析揭示意大利蜜蜂响应东方蜜蜂微孢子虫胁迫的免疫应答机制

Unraveling the mechanism underlying the immune responses of Apis mellifera ligustica to Nosema ceranae stress based on small RNA omics analyses

摘要:

[目的] 通过深度测序和组学分析在small RNA组学层面揭示意大利蜜蜂（Apis mellifera ligustica，简称意蜂）响应东方蜜蜂微孢子虫（Nosema ceranae）的免疫应答机制。[方法] 利用small RNA-seq技术对正常及N. ceranae胁迫7 d和10 d的意蜂工蜂中肠（Am7CK、Am7T、Am10CK和Am10T）进行深度测序。利用相关生物信息学软件对测序数据进行质控、已知microRNA（miRNA）鉴定、新miRNA预测及miRNA的结构特征分析。通过Stem-loop RT-PCR验证新miRNA的表达。按照|log₂（Fold change）|≥ 1和P ≤ 0.05的标准筛选出Am7CKvs.Am7T和Am10CK vs.Am10T比较组的差异表达miRNA（differentially expressed miRNA，DEmiRNA）。利用软件预测DEmiRNA靶向结合的mRNA并进行GO和KEGG数据库注释，根据注释信息对细胞和体液免疫相关通路及富集靶mRNA进行统计和分析。根据靶向结合关系构建DEmiRNA和免疫通路相关差异表达mRNA（differentially expressed mRNA，DEmRNA）的调控网络。采用RT-qPCR对数据的可靠性和DEmiRNA的差异表达进行验证。[结果] 共获得165895574条原始读段和132028990条有效序列标签，各组的组内Pearson相关性平均在87.92%及以上。共鉴定到928个已知miRNA和56个新miRNA。这些miRNA的长度介于18-28 nt，多数的长度为18 nt和22 nt且首位碱基主要偏向U。验证了12个新miRNA的真实表达。Am7CKvs.Am7T比较组包含48个上调miRNA和36个下调miRNA；Am10CKvs.Am10T比较组包含56个上调miRNA和51个下调miRNA。两个比较组的DEmiRNA可分别靶向结合9827个和10720个mRNA。这些靶mRNA可分别注释到50和47条功能条目，以及138和135条KEGG通路。DEmiRNA与免疫通路相关靶mRNA的调控网络分析结果显示，Am7CKvs.Am7T中有26个DEmiRNA靶向与内吞作用等免疫通路相关的10个DEmRNA；Am10CKvs.Am10T中有15个DEmiRNA靶向与MAPK信号通路等免疫通路相关的10个DEmRNA。验证了测序数据和4个DEmiRNA差异表达的可靠性。[结论] 研究结果揭示了宿主DEmiRNA可能通过调控物质和能量代谢、细胞和体液免疫对N. ceranae产生应答，但DEmiRNA不参与抗菌肽基因的表达调控；miR-1-z可能参与宿主的细胞增殖、细胞凋亡和免疫进程；氧化磷酸化通路可能在宿主免疫应答及宿主-病原互作中发挥特殊作用。

Abstract:

[Objective] To reveal the mechanism underlying immune responses of Apis mellifera ligustica to Nosema ceranae stress at small RNA transcriptome level based on deep sequencing and omics analysis. [Methods] A. m. ligustica workers' midguts at 7 and 10 days post N. ceranae stress (Am7T, Am10T) and corresponding normal midguts (Am7CK, Am10CK) were sequenced using small RNA-seq. Related bioinformatic softwares were used to perform quality control of sequencing data, identification of known miRNAs and novel miRNAs and analysis of structural features of miRNAs. The expression of novel miRNAs was verified by Stem-loop RT-PCR. Differentially expressed miRNAs (DEmiRNAs) in Am7CK vs. Am7T and Am10CK vs. Am10T comparison groups were screened out following the standards of|log₂(Fold change)| ≥ 1 and P ≤ 0.05. Target mRNAs of DEmiRNAs were predicted followed by annotation in GO and KEGG databases using softwares, based on annotation information, summary and investigation of cellular and humoral immune-associated pathways and enriched target mRNAs were conducted. Regulatory networks of DEmiRNAs and differentially expressed mRNAs (DEmRNAs) related to immune pathways were constructed according to target binding relationship. RT-qPCR was performed to validate the sequencing data and differential expression of DEmiRNAs. [Results] Here, 165895574 raw reads and 132028990 clean tags were yielded, and average Pearson correlation coefficients among different biological replicas in every group were above 87.92%. 928 known miRNAs and 56 novel miRNAs were identified. The length of these miRNAs was among 18-28 nt, with the most abundant length 18 nt and 22 nt; the first base of most miRNAs had a U bias. The true expression of 12 novel miRNAs was verified. 48 up-regulated miRNAs and 36 down-regulated miRNAs were identified in Am7CK vs. Am7T, while 56 up-regulated miRNAs and 51 down-regulated miRNAs were identified in Am10CK vs. Am10T. These DEmiRNAs can respectively target 9827 and 10720 mRNAs, involving in 50 and 47 functional terms and 138 and 135 pathways. Analysis of regulatory networks of DEmiRNAs and target mRNAs related to immune pathways showed 26 DEmiRNAs in Am7CK vs. Am7T could target 10 DEmRNAs such as endocytosis, whereas 15 DEmiRNAs in Am10CK vs. Am10T can target 10 immune-associated pathways including MAPK signaling pathway. The reliability of sequencing data and differential expression trend of four DEmiRNA was validated. [Conclusion] Host DEmiRNAs may response to N. ceranae through regulating material and energy metabolisms, as well as cellular and humoral immune, but might not regulate the expression of antimicrobial peptide-encoded genes; miR-1-z was likely to participate in cell proliferation, apoptosis and immune processes; oxidative phosphorylation pathway may play a special role in host immune response and host-pathogen interaction.