蜜蜂球囊菌菌丝和孢子中长链非编码RNA的比较及潜在功能分析  

Comparison and Potential Functional Analysis of Long Non-Coding RNAs Between Ascosphaera apis Mycelium and Spore

作　　者：陈华枝 王杰 祝智威 蒋海宾 范元婵 范小雪 万洁琦 卢家轩 郑燕珍 付中民 徐国钧[1] 陈大福 郭睿 CHEN HuaZhi;WANG Jie;ZHU ZhiWei;JIANG HaiBin;FAN YuanChan;FAN XiaoXue;WAN JieQi;LU JiaXuan;ZHENG YanZhen;FU ZhongMin;XU GuoJun;CHEN DaFu;GUO Rui(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002;Apitherapy Research Institution,Fujian Agriculture and Forestry University,Fuzhou 350002;Engineering Research Center of Processing and Application of Bee Products of Ministry of Education,Fujian Agriculture and Forestry University,Fuzhou 350002)

机构地区：[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 [3]福建农林大学蜂产品加工与应用教育部工程研究中心,福州350002

出　　处：《中国农业科学》2021年第2期435-448,共14页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31702190);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);福建农林大学硕士生导师团队(郭睿);福建农林大学科技创新专项基金(CXZX2017342,CXZX2017343);福建省大学生创新创业项目(202010389016,202010389162)。

摘　　要：【背景】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,可引起成年蜜蜂数量和蜂群群势的急剧下降。长链非编码RNA(long non-coding RNA,lncRNA)是一类新近发现的非编码RNA,在表观遗传、细胞周期、剂量补偿等众多生命活动中发挥重要生物学功能。【目的】明确球囊菌菌丝和孢子中lncRNA的数量、种类和表达谱差异,并探究共有lncRNA、特有lncRNA和差异表达lncRNA(differentially expressed lncRNA,DElncRNA)在菌丝与孢子中的潜在功能。【方法】利用基于链特异性建库的lncRNA-seq技术对球囊菌的纯化菌丝(AaM)和纯化孢子(AaS)分别进行测序。根据FPKM(Fragment Per Kilobase of per Million mapped reads)法计算lncRNA在AaM和AaS中的表达水平。通过Venn分析筛选AaM与AaS的共有lncRNA和特有lncRNA。按照P≤0.05且|log2 fold change|≥1的标准筛选AaM vs AaS比较组中的DElncRNA。通过Blast工具将共有lncRNA、特有lncRNA和DElncRNA的上下游基因比对GO和KEGG数据库,以进行功能及通路注释。根据靶向结合关系构建共有lncRNA、特有lncRNA和DElncRNA的竞争性内源RNA(competing endogenous RNA,ceRNA)调控网络并利用Cytoscape软件进行可视化。利用RT-qPCR验证测序数据的可靠性。【结果】AaM和AaS分别测得108614646和105675408条原始读段(raw reads),经严格过滤得到107780032和104621402条有效读段(clean reads),Q20分别为98.76%和98.72%,Q30分别达到95.84%和95.78%。共鉴定到850个lncRNA。AaM和AaS的共有lncRNA为701个,二者的特有lncRNA分别为39和110个。上述共有lncRNA通过顺式作用调控3992个上下游基因,它们涉及细胞进程、代谢进程和催化活性等42个功能条目,以及代谢途径、次生代谢物的生物合成和抗生素的生物合成等117条通路;AaM的特有lncRNA和AaS的特有lncRNA通过顺式作用分别调控243和672个上下游基因。AaM vs AaS比较组包含的255个DElncRNA通过顺式作用调控1【Background】Ascosphaera apis is a fungal pathogen that exclusively infects honeybee larvae,causing a sharp decrease of the population of adult honeybees and colony population.Long non-coding RNA(lncRNA),a recently discovered non-coding RNA(ncRNA),plays a vital biological role in various activities such as epigenetics,cell cycle and dose compensation.【Objective】This study aimed to clarify the differences of number,type and expression profile of lncRNAs between A.apis mycelium and spore,and investigate the potential role of the common lncRNAs,specific lncRNAs and differentially expressed lncRNAs(DElncRNAs).【Method】The purified mycelia(AaM)and purified spores(AaS)of A.apis were respectively sequenced using strand specific library-based lncRNA-seq technology.The expression levels of lncRNAs in AaM and AaS were calculated using FPKM(Fragment Per Kilobase of per Million mapped reads)method.Common lncRNAs and specific lncRNAs were filtered out following Venn analysis.DElncRNAs within AaM vs Aa S comparison group were screened out following the standard of P≤0.05 and|log2 fold change|≥1.Upstream and downstream genes of common lncRNAs,specific lncRNAs and DElncRNAs were aligned against GO and KEGG databases to obtain function and pathway annotations.The competing endogenous RNA(ce RNA)regulation networks of common lncRNAs,specific lncRNAs and DElncRNAs were constructed following target binding relationships,followed by visualization using Cytoscape software.RT-qPCR was performed to verify the reliability of the sequencing data.【Result】In total,108614646 and 105675408 raw reads were gained from AaM and AaS,and after strict filtering,107780032 and 104621402 clean reads were obtained,respectively,with Q20 of 98.76%and 98.72%,and Q30 of 95.84%and 95.78%.A total of 850 lncRNAs were identified.Seven hundred and one lncRNAs were shared by AaM and AaS,and there were 39 and 110 specific lncRNAs.Via cis function,these shared lncRNAs could regulate 3992 upstream and downstream genes involving in 42 functional ter

关 键 词：蜜蜂球囊菌 长链非编码RNA 菌丝 孢子 竞争性内源RNA 

分 类 号：S895.137[农业科学—特种经济动物饲养]