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

【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micro RNA(mi RNA)鉴定、新mi RNA预测及mi RNA的结构特征分析。通过Stem-loop RT-PCR验证新mi RNA的表达。按照|log2(Fold change)|≥1和P≤0.05的标准筛选出Am7CKvs.Am7T和Am10CKvs.Am10T比较组的差异表达mi RNA(differentially expressed mi RNA,DEmi RNA)。利用软件预测DEmi RNA靶向结合的m RNA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m RNA进行统计和分析。根据靶向结合关系构建DEmi RNA和免疫通路相关差异表达m RNA(differentially expressed m RNA,DEm RNA)的调控网络。采用RT-q PCR对数据的可靠性和DEmi RNA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mi RNA和56个新mi RNA。这些mi RNA的长度介于18–28nt,多数的长度为18nt和22nt且首位碱基主要偏向U。验证了12个新mi RNA的真实表达。Am7CKvs.Am7T比较组包含48个上调mi RNA和36个下调mi RNA;Am10CK vs.Am10T比较组包含56个上调mi RNA和51个下调mi RNA。两个比较组的DEmi RNA可分别靶向结合9827个和10720个m RNA。这些靶m RNA可分别注释到50和47条功能条目,以及138和135条KEGG通路。DEmi RNA与免疫通路相关靶m RNA的调控网络分析结果显示,Am7CK vs.Am7T中有26个DEmi RNA靶向与内吞作用等免疫通路相关的10个DEm RNA;Am10CK vs.Am10T中有15个DEmi RNA靶向与MAPK信号通路等免疫通路相关的10个DEm RNA。验证了测序数据和4个DEmi RNA差异表达的可靠性。【结论】研究结果揭示了宿主DEmi RNA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DEmi RNA不参与抗菌肽基因的表达调控;mi R-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。

[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(Am7 T,Am10 T)and corresponding normal midguts(Am7 CK,Am10 CK)were sequenced using small RNA-seq.Related bioinformatic softwares were used to perform quality control of sequencing data,identification of known mi RNAs and novel mi RNAs and analysis of structural features of mi RNAs.The expression of novel mi RNAs was verified by Stem-loop RT-PCR.Differentially expressed mi RNAs(DEmi RNAs)in Am7 CK vs.Am7 T and Am10 CK vs.Am10 T comparison groups were screened out following the standards of|log2(Fold change)|≥1 and P≤0.05.Target mR NAs of DEmi RNAs 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 mR NAs were conducted.Regulatory networks of DEmi RNAs and differentially expressed mR NAs(DEmR NAs)related to immune pathways were constructed according to target binding relationship.RT-q PCR was performed to validate the sequencing data and differential expression of DEmi RNAs.[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 mi RNAs and 56 novel mi RNAs were identified.The length of these mi RNAs was among 18–28 nt,with the most abundant length 18 nt and 22 nt;the first base of most mi RNAs had a U bias.The true expression of 12 novel mi RNAs was verified.48 up-regulated mi RNAs and 36 down-regulated mi RNAs were identified in Am7 CK vs.Am7 T,while 56 up-regulated mi RNAs and 51 down-regulated mi RNAs were identified in Am10 CK vs.Am10 T.These DEmi RNAs can respectively target 9827 and 10720 m RNAs,involving in 50 and 47 functional terms and 138 and 135 pathways.Analysis of regulatory networks of DEmi RNAs and target mR NAs related to immune pathways showed 26 DEmi RNAs in Am7 CK vs.Am7 T could target 10 DEm RNAs such as endocytosis,whereas 15 DEmi RNAs in Am10 CK vs.Am10 T can target 10 immune-associated pathways including MAPK signaling pathway.The reliability of sequencing data and differential expression trend of four DEmi RNA was validated.[Conclusion]Host DEmi RNAs 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;mi R-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.