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

作　　者：杜宇 王杰 蒋海宾 王秀娜[3,4] 范元婵 范小雪 祝智威 隆琦 张文德 熊翠玲 郑燕珍 付中民 陈大福 郭睿[1] Yu Du;Jie Wang;Haibin Jiang;Xiuna Wang;Yuanchan Fan;Xiaoxue Fan;Zhiwei Zhu;Qi Long;Wende Zhang;Cuiling Xiong;Yanzhen Zheng;Zhongmin Fu;Dafu Chen;Rui Guo(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian Province,China;Engineering Research Center of Processing and Application of Bee Products of Ministry of Education,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian Province,China;College of Life Sciences,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian Province,China;Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province,Fuzhou 350002,Fujian Province,China)

机构地区：[1]福建农林大学动物科学学院(蜂学学院),福建福州350002 [2]福建农林大学蜂产品加工与应用教育部工程研究中心,福建福州350002 [3]福建农林大学生命科学学院,福建福州350002 [4]福建省病原真菌与真菌毒素重点实验室,福建福州350002

出　　处：《微生物学报》2021年第3期667-682,共16页Acta Microbiologica Sinica

基　　金：国家现代农业产业技术体系建设专项资金(CARS-44-KXJ7);福建省自然科学基金(2018J05042);福建省教育厅中青年教师教育科研项目(JAT170158);福建农林大学硕士生导师团队项目(郭睿);福建农林大学杰出青年科研人才计划(xjq201814);福建农林大学优秀硕士学位论文资助基金(杜宇);福建省病原真菌与真菌毒素重点实验室(福建农林大学)开放课题。

摘　　要：【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,[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 ident

关 键 词：长读段测序 牛津纳米孔 蜜蜂球囊菌 可变剪切 可变腺苷酸化 

分 类 号：S895.137[农业科学—特种经济动物饲养]