基于第三代长读段测序数据解析蜜蜂球囊菌基因的可变剪切与可变腺苷酸化

Analysis of alternative splicing and polyadenylation of Ascosphaera apis genes based on third-generation long-read sequencing dataset

【目的】蜜蜂球囊菌(Ascosphaera apis,简称球囊菌)是一种专性侵染蜜蜂幼虫的真菌病原,导致的白垩病是严重影响养蜂生产的顽疾,每年给养蜂业造成较大损失。本研究旨在基于已获得的第三代长读段测序数据对球囊菌菌丝(Aam)和孢子(Aas)中基因的可变剪切(alternative splicing,AS)和可变多聚腺苷酸化(alternative polyadenylation,APA)进行深入分析。【方法】利用Astalavista软件鉴定Aam和Aas中基因的AS事件类型。利用IGV浏览器对部分剪切异构体(isoform)的结构进行可视化。通过TAPIS pipeline对Aam和Aas中基因的APA位点进行鉴定。通过MEME软件对Aam和Aas中全长转录本的APA位点上游50bp的序列特征进行分析并对motif进行鉴定。【结果】在Aam中共鉴定到286次AS事件,包括162次RI(Retained intron),87次A3(Alternative 3'splice-site)、32次A5(Alternative 5'splice-site)和5次SE (Skipping exon);在Aas中共鉴定到559次AS事件,包括305次RI、155次A3、85次A5、13次SE和1次MEE (Mutually exclusive exon)。进一步分析发现,现有参考基因组上的多数注释基因结构并不完整;部分注释基因在菌丝和孢子中转录形成的isoform在数量和结构方面均存在差异;部分isoform在参考基因组中没有对应的注释基因。Aam中共鉴定到2748个基因含有1个及以上的APA位点,其中含有1个APA位点的基因数量最多(726,26.42%);Aas中共鉴定到2768个基因含有1个及以上的APA位点,其中含有5个以上APA位点的基因数量最多(1180,42.63%)。部分基因在菌丝和孢子中含有不同的APA位点数。序列特征分析结果显示,球囊菌全长转录本的3'UTR的上下游表现出明显的碱基倾向性,U和A分别富集在3'UTR的上游和下游。此外,在球囊菌全长转录本的APA位点上游鉴定到4个motif,分别是UCUCCU、UCUUCU、CCCACC和CCCCCU。【结论】本研究通过对球囊菌菌丝和孢子中基因的AS和APA进行深入分析,揭示了球囊菌转录组的复杂性,为完善现有的基因组和转录组注释提供了宝贵信息,也为探究AS和APA在球囊菌的基因表达调控中的作用提供了关键基础。

[Objective]Ascosphaera apis is a fungal pathogen that exclusively infects honeybee larvae,leading to chalkbrood,which is a chronic disease in beekeeping industry and results in heavy losses for apiculture.The objective of this work is to investigate alternative splicing(AS)and alternative polyadenylation(APA)of genes in A.apis mycelium(Aam)and spore(Aas)based on previously gained third-generation long-read sequencing dataset.[Methods]The type of AS events occurred in genes in Aam and Aas was identified.The visualization of partial isoforms’structures was performed with IGV browser.APA sites of genes in Aam and Aas were identified using TAPIS pipeline.MEME software was used to investigate characteristics of sequences at 50 bp upstream of APA sites followed by identification of motifs.[Results]In total,286 AS events were identified in Aam,including 162 retained intron(RI),87 alternative 3’splice-site(A3),32 alternative 5’splice-site(A5)and five skipping exon(SE),while 559 AS events were identified in Aas,including 305 RI,155 A3,85 A5,13 SE and one mutually exclusive exon(MEE).Further analysis suggested that majority of annotated genes in current reference genome are incomplete,number and structure of partial annotated genes in mycelium differ from those in spore,and for part of isoforms,there are no corresponding annotated genes in reference genome.Additionally,a total of 2748 genes in Aam were observed to contain one and more APA sites,among them those containing one APA site was the largest group(726,26.42%);while in Aas 2768 gene were found to contain one and more APA sites,and those containing more than five APA sites were the most abundant(1180,42.63%).Besides,part of genes in A.apis mycelium and spore were detected to have various APA sites.Moreover,analysis of sequence characteristics indicated that upstream and downstream sequences of 3’UTR of A.apis full-length transcripts have an obvious base bias,and U and A were respectively enriched in the upstream and downstream.Moreover,four motifs were identified at the upstream of APA sites of A.apis full-length transcripts,including UCUCCU,UCUUCU,CCCACC and CCCCCU.[Conclusion]In this study,AS and APA of genes in A.apis mycelium and spore were deeply analyzed,the results uncovered the complexity of A.apis transcriptome,offering valuable information for improvement of current genome and transcriptome annotations and a pivotal foundation for exploration of the function of AS and APA involved in regulation of A.apis gene expression.