东方蜜蜂微孢子虫孢子中微小RNA的鉴定与分析

【目的】丰富东方蜜蜂微孢子虫Nosema ceranae的微小RNA(microRNA, miRNA)信息，并为深入探究miRNA在病原孢子和病原侵染中的功能提供理论和实验依据。【方法】基于已获得的small RNA-seq数据，利用生物信息学软件对东方蜜蜂微孢子虫的纯净孢子中的miRNA进行鉴定和分析。采用茎环反转录PCR(stem-loop RT-PCR)检测已鉴定的miRNA的表达；通过分子克隆与Sanger测序验证miRNA的序列。使用TargetFinder软件预测这些miRNA的靶基因，并对靶基因进行数据库注释。根据miRNA与靶基因的靶向结合关系构建调控网络，再利用Cytoscape软件进行可视化。【结果】在东方蜜蜂微孢子虫孢子中共鉴定到10个miRNA；这些miRNA的长度分布介于21~25 nt，首位碱基表现出U偏向性，每一位碱基的偏向性差异明显。Stem-loop RT-PCR检测结果表明这10个miRNA均真实表达；Sanger测序结果证实了随机选取的其中2个miRNA的序列真实性。共预测出249个靶基因，其中分别有249, 118, 136和3个靶基因可注释到Nr,Swiss-Prot, KOG和eggNOG数据库。此外，分别有134和71个靶基因可分别注释到GO数据库的30个功能条目和KEGG数据库的54条通路。【结论】本研究揭示了东方蜜蜂微孢子虫孢子中miRNA的存在和表达；这些miRNA通过调控潜在靶基因的表达参与孢子的生命活动。

Identification and analysis of microRNAs inNosema ceranaespores

【Aim】 To enrich the information of microRNA (miRNA) in Nosema ceranae and toprovide theoretical and experimental bases for further exploration of the function ofmiRNAs in pathogen spore and pathogen infection. 【Methods】 Based on the gained data fromsmall RNA-seq, miRNAs in the clean spores of N. ceranae were identified and analyzed usingbioinformatics software. Stem-loop RT-PCR was performed to detect the expression of theidentified miRNAs. Sequences of miRNAs were validated by molecular cloning and Sangersequencing. Target genes of these miRNAs were predicted with TargetFinder software and thenannotated to databases. The regulatory network was constructed based on the the targetingrelationship between miRNAs and their target genes, followed by visualization withCytoscape software. 【Results】 In total, 10 miRNAs were identified in N. ceranae spores.The length distribution of these miRNAs ranged from 21 to 25 nt. The first base of thesemiRNAs showed a U bias, and there was an obvious difference in the bias at each base. Theresult of stem-loop RT-PCR indicated that the 10 miRNAs were truly expressed, and Sangersequencing confirmed the reliability of sequences of two miRNAs randomly selected fromthem. A total of 249 target genes were predicted, among which 249, 118, 136 and 3 targetgenes could be annotated to Nr, Swiss-Prot, KOG and eggNOG databases, respectively. Inaddition, 134 and 71 target genes could be respectively annotated to 30 functional terms inGO database and 54 pathways in KEGG database. 【Conclusion】 The findings reveal thepresence and expression of miRNAs in N. ceranae spores. These miRNAs may participate invital activities in spores via regulating the expression of potential target genes.