东方蜜蜂微孢子虫孢子中微小RNA的鉴定与分析

doi:10.16380/j.kcxb.2022.06.006

昆虫学报 ›› 2022, Vol. 65 ›› Issue (6): 708-717.doi: 10.16380/j.kcxb.2022.06.006

东方蜜蜂微孢子虫孢子中微小RNA的鉴定与分析

张文德^1,#, 赵浩东^1,#, 孙明会¹, 余岢骏¹, 郭意龙¹, 朱乐冉¹, 胡颖¹, 赵萧¹, 叶亚萍¹, 陈大福^1,2,*, 郭睿^1,2,*

(1. 福建农林大学动物科学学院(蜂学学院),福州 350002; 2. 福建农林大学蜂疗研究所, 福州 350002)

出版日期:2022-06-20 发布日期:2022-07-08

Identification and analysis of microRNAs in Nosema ceranae spores

ZHANG Wen-De^1,#, ZHAO Hao-Dong^1,#, SUN Ming-Hui¹, YU Ke-Jun¹, GUO Yi-Long¹, ZHU Le-Ran¹, HU Ying¹, ZHAO Xiao¹, YE Ya-Ping¹, CHEN Da-Fu^1,2,*, GUO Rui^1,2,*

(1. College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2. Apitherapy Research Institute, Fujian Agriculture and Forestry University, Fuzhou 350002, China)

Online:2022-06-20 Published:2022-07-08

摘要/Abstract

摘要： 【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。

关键词: 蜜蜂, 宿主, 东方蜜蜂微孢子虫, 小RNA测序, 微小RNA, 调控

Abstract: 【Aim】 To enrich the information of microRNA (miRNA) in Nosema ceranae and to provide theoretical and experimental bases for further exploration of the function of miRNAs in pathogen spore and pathogen infection. 【Methods】 Based on the gained data from small RNA-seq, miRNAs in the clean spores of N. ceranae were identified and analyzed using bioinformatics software. Stem-loop RT-PCR was performed to detect the expression of the identified miRNAs. Sequences of miRNAs were validated by molecular cloning and Sanger sequencing. Target genes of these miRNAs were predicted with TargetFinder software and then annotated to databases. The regulatory network was constructed based on the the targeting relationship between miRNAs and their target genes, followed by visualization with Cytoscape 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 these miRNAs showed a U bias, and there was an obvious difference in the bias at each base. The result of stem-loop RT-PCR indicated that the 10 miRNAs were truly expressed, and Sanger sequencing confirmed the reliability of sequences of two miRNAs randomly selected from them. A total of 249 target genes were predicted, among which 249, 118, 136 and 3 target genes could be annotated to Nr, Swiss-Prot, KOG and eggNOG databases, respectively. In addition, 134 and 71 target genes could be respectively annotated to 30 functional terms in GO database and 54 pathways in KEGG database. 【Conclusion】 The findings reveal the presence and expression of miRNAs in N. ceranae spores. These miRNAs may participate in vital activities in spores via regulating the expression of potential target genes.

Key words: Honey bee, host, Nosema ceranae, small RNA sequencing, microRNA, regulation

张文德, 赵浩东, 孙明会, 余岢骏, 郭意龙, 朱乐冉, 胡颖, 赵萧, 叶亚萍, 陈大福, 郭睿. 东方蜜蜂微孢子虫孢子中微小RNA的鉴定与分析[J]. 昆虫学报, 2022, 65(6): 708-717.

ZHANG Wen-De, ZHAO Hao-Dong, SUN Ming-Hui, YU Ke-Jun, GUO Yi-Long, ZHU Le-Ran, HU Ying, ZHAO Xiao, YE Ya-Ping, CHEN Da-Fu, GUO Rui. Identification and analysis of microRNAs in Nosema ceranae spores[J]. Acta Entomologica Sinica, 2022, 65(6): 708-717.

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东方蜜蜂微孢子虫孢子中微小RNA的鉴定与分析

Identification and analysis of microRNAs in Nosema ceranae spores

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