蜜蜂球囊菌的microRNA鉴定及其调控网络分析

Identification of Ascosphaera apis microRNAs and investigation of their regulation networks

摘要:

[目的]本研究利用small RNA-seq技术对球囊菌的纯培养进行测序，对球囊菌的microRNAs miRNAs）进行预测、鉴定和分析，进而构建miRNAs-mRNAs的调控网络。[方法]利用Illumina Hiseq Xten平台对球囊菌菌丝与孢子进行测序，通过相关生物信息学软件对球囊菌的miRNAs进行预测和分析，通过茎环（Stem-loop） PCR对部分miRNAs进行鉴定，利用Cytoskype软件构建miRNAs-mRNAs的调控网络。[结果]本研究共获得48268696条clean reads，预测出118个球囊菌的miRNAs，它们的长度分布介于18–25 nt之间，不同长度的miRNA的首位碱基偏好性差异明显。Stem-loop PCR验证结果显示共有10个miRNAs能够扩增出符合预期的目的片段，说明多数miRNAs可能真实存在。共预测出6529个球囊菌miRNAs的靶基因，其中5725个能够注释到Nr、Swissprot、KOG、GO和KEGG数据库。进一步分析结果显示有24个靶基因注释在MAPK信号通路。Cytoskype软件分析结果显示球囊菌的miRNAs与mRNAs之间存在复杂的调控网络，绝大多数的miRNAs处于调控网络的内部且同时结合多个mRNAs。[结论]本研究率先对球囊菌的miRNAs及miRNAs-mRNAs调控网络进行全面分析，研究结果丰富了对球囊菌miRNAs的认识，为其基础生物学信息提供了有益补充，也为阐明球囊菌致病的分子机理打下了一定基础。

Abstract:

[Objective] In this study, pure culture of A. apis was sequenced using sRNA-seq technology, followed by prediction, identification and analysis of A. apis microRNAs. The microRNAs-mRNAs regulation network was further constructed. [Methods] Illumina Hiseq Xten platform was used to sequence mycelium and spores of A. apis, and corresponding softwares were used to predict and analyze A. apis microRNAs, some of which were identified via Stem-loop PCR. Cytoskype software was used to construct A. apis microRNAs-mRNAs regulation network. [Results] A total of 48268696 clean reads were obtained, and 118 miRNAs of A. apis were predicted, whose length was distributed between 18 nt and 25 nt. The preference of the first base of miRNAs with different length was obviously various. Stem-loop PCR result showed target fragments with expected sizes were amplified from 10 microRNAs, implying most of the predicted microRNAs' true existence. In total, 6529 target genes of A. apis microRNAs were predicted, and among them 5725 could be annotated in Nr, Swissprot, KOG, GO and KEGG databases. Further investigation demonstrated 24 target genes were annotated in MAPK signaling pathway. Cytoskype software analysis suggested complicated regulation networks exist between microRNAs and mRNAs in A. apis, and majority of miRNAs inside the networks bind to several mRNAs. [Conclusion] Our findings enrich the understanding of A. apis microRNAs, provide beneficial supplement for basic biology information of A. apis, and lay some foundation for illustrating the molecular mechanism regulating the pathogenesis of this widespread fungal pathogen.