意大利蜜蜂工蜂中肠发育过程中长链非编码RNA的差异表达分析  

Differential Expression Analysis of Long Non-Coding RNAs During the Developmental Process of Apis mellifera ligustica Worker's Midgut

作　　者：郭睿[1] 耿四海 熊翠玲[1] 郑燕珍[1] 付中民[1] 王海朋 杜宇[1] 童新宇 赵红霞 陈大福[1] GUO Rui;GENG SiHai;XIONG CuiLing;ZHENG YanZhen;FU ZhongMin;WANG HaiPeng;DU Yu;TONG XinYu;ZHAO HongXia;CHEN DaFu(College of Bee Science,Fujian Agriculture and Forestry University,Fuzhou 350002;Guangdong Institute of Applied Biological Resources,Guangzhou 510260)

机构地区：[1]福建农林大学蜂学学院,福州350002 [2]广东省生物资源应用研究所,广州510260

出　　处：《中国农业科学》2018年第18期3600-3613,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);福建省教育厅中青年教师教育科研项目(JAT170158);福建农林大学科技创新专项基金(CXZX2017343);福建省大学生创新创业训练计划(201610389053)

摘　　要：【目的】长链非编码RNA(lncRNA)在真核生物的基因表达、表观遗传和细胞周期调控等方面发挥重要功能。本研究旨在探究意大利蜜蜂(Apis mellifera ligustica,简称意蜂)工蜂中肠发育过程中lncRNA的表达谱及其作用。【方法】利用RNA-seq技术和链特异性建库方法对意蜂7和10日龄工蜂中肠(Am7、Am10)进行深度测序,下机的原始数据经过Perl脚本过滤得到高质量有效读段。利用bowtie工具将有效读段比对核糖体数据库,进一步利用Top Hat2软件将未比对到核糖体数据库上的数据比对到参考基因组。利用CPC和CNCI软件对转录本的编码能力进行预测。通过RT-PCR对部分lncRNA进行鉴定。利用edgeR软件进行差异表达lncRNA(DElncRNA)分析,进而预测lncRNA的上下游基因,并对上下游基因进行GO及KEGG代谢通路富集分析。联用RNAhybrid、Miranda和Target Scan软件预测DElncRNA靶向结合的mi RNA及mi RNA靶向结合的靶基因,并通过Cytoscape软件构建DElncRNAs-mi RNAs-m RNAs的调控网络。最后,通过RT-qPCR验证测序数据的可靠性。【结果】Am7和Am10的深度测序分别获得134 802 058和147 051 470条原始读段,经严格过滤分别得到134 166 157和146 293 288条有效读段;共得到3 890个DElncRNA,包括2 005个上调lncRNA与1 885个下调lncRNA。RT-PCR验证结果显示共有8个lncRNA能扩增出符合预期的目的片段,表明预测出的lncRNA真实存在。DElncRNA的上下游基因数为1 793个,它们涉及42个GO条目,包括代谢进程、发育进程、细胞进程、应激反应和免疫系统进程等;这些上下游基因还涉及251条代谢通路,包括碳代谢、嘌呤代谢和脂肪酸的生物合成等物质代谢通路,硫代谢、甲烷代谢和氧化磷酸化等能量代谢通路,Hippo信号通路、Wnt信号通路和Notch信号通路等信号通路,溶酶体、内吞作用和泛素介导的蛋白水解等细胞免疫通路,以及MAPK信号通路、Jak-STAT信号通路和NF-kappa B信号通路等【Objective】Long non-coding RNA(lncRNA)plays an important role in regulation of gene expression,epigenetics and cell cycle in eukaryotes.The objective of this study is to investigate the expression profile and role of lncRNAs in the developmental process of Apis mellifera ligustica worker’s midgut.【Method】In this study,7-and 10-day-old worker’s midguts of A.m.ligustica(Am7,Am10)were sequenced using RNA-seq technology and strand-specific library construction method.Using Perl script,raw reads were filtered to obtain clean reads with high-quality.Bowtie tool was used to compare clean reads to the ribosome database,and TopHat2 software was employed to compare unmapped clean reads to the reference genome.CPC and CNCI softwares were utilized to predict coding capacity of the transcripts.RT-PCR was performed to identify partial lncRNAs.Investigation of differentially expressed lncRNAs(DElncRNAs)was carried out with edgeR,followed by prediction of upstream and downstream genes,for which GO and KEGG pathway enrichment analyses were performed.RNAhybrid,Miranda and TargetScan softwares were utilized together to predict target miRNAs of DElncRNAs and target genes of miRNAs,and DElncRNAs-miRNAs-mRNAs regulation networks were visualized via Cytoscape.Finally,RT-qPCR was conducted to verify reliability of the sequencing data.【Result】134 802 058 and 147 051 470 raw reads were gained from deep sequencing of Am7 and Am10,respectively,and after stringent filtration,134 166 157 and 146 293 288 were obtained.In total,6 353 lncRNAs were predicted,and 3 890 DElncRNAs were obtained based on expression calculation,including 2 005 up-regulated lncRNAs and 1 885 down-regulated lncRNAs.The result of RT-PCR suggested the expected signal bands could be amplified from 8 lncRNAs,implying their true existence.There were 1 793 upstream and downstream genes of DElncRNAs,which were involved in 42 GO terms,including metabolic processes,developmental processes,cellular processes,stress responses,immune system processes and so forth.Th

关 键 词：意大利蜜蜂 中肠 发育 长链非编码RNA 上下游基因 

分 类 号：S891[农业科学—特种经济动物饲养]