意大利蜜蜂工蜂中肠发育过程中的差异基因表达谱及调控网络  

Profiling and Regulation Network of Differentially Expressed Genes During the Development Process of Apis mellifera ligustica Worker’s Midgut

作　　者：杜宇 周丁丁 万洁琦 卢家轩 范小雪 范元婵 陈恒 熊翠玲 郑燕珍 付中民 徐国钧 陈大福 郭睿 DU Yu;ZHOU DingDing;WAN JieQi;LU JiaXuan;FAN XiaoXue;FAN YuanChan;CHEN Heng;XIONG CuiLing;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)

机构地区：[1]福建农林大学动物科学学院(蜂学学院)

出　　处：《中国农业科学》2020年第1期201-212,共12页Scientia Agricultura 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);福建农林大学杰出青年科研人才计划(xjq201814);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训练计划(3165602032,3155006018)

摘　　要：【目的】前期已对意大利蜜蜂(Apis mellifera ligustica,简称意蜂)7日龄工蜂中肠(Am7)、10日龄工蜂中肠(Am10)进行全转录组测序,本研究基于高质量的组学数据探究中肠发育过程中的差异基因表达谱及其调控网络,以期解析意蜂工蜂中肠发育的分子机理。【方法】根据FPKM(fragments per kilobase of transcript per million mapped reads)算法计算基因表达量,并以|log2 fold change|≥1且P≤0.05作为标准筛选得到差异表达基因(differentially expressed gene,DEG)。利用TargetFinder软件预测ame-miR-6001-3p的靶mRNA。利用相关生物信息学软件,对全部DEG进行GO和KEGG数据库注释。筛选出与AMPK、P13K-Akt、Wnt、cAMP、FoxO、Hippo、mTOR、Jak-STAT、Toll-like受体、TGF-beta、Notch、MAPK和NF-κB 13条信号通路存在富集关系的DEG,以及与ame-miR-6001-3p存在靶向结合关系的DEG,通过Cytoscape软件构建调控网络将其富集关系与调控关系可视化。利用茎环反转录PCR(Stem loop RT-PCR)和实时荧光定量PCR(RT-qPCR)验证ame-miR-6001-3p以及DEG在Am7和Am10中的差异表达情况。【结果】Am7 vs Am10比较组中共有1 038个DEG,包括515个上调基因和523个下调基因。这些DEG涉及细胞进程、代谢进程和催化活性等功能条目,并显著富集在氧化磷酸化、氨基糖与核苷酸糖代谢、脂肪酸代谢和嘌呤代谢等能量和物质代谢通路,表明工蜂中肠内存在旺盛细胞生命与新陈代谢活动。表达量聚类分析发现分别有20、18、15和14个DEG富集在AMPK信号通路、P13K-Akt信号通路、内吞作用和Hippo信号通路。57个DEG与P13K-Akt、Wnt、Jak-STAT等上述13条与生长发育和免疫防御相关的信号通路存在富集关系,且1个DEG可与多条信号通路存在富集关系。调控网络分析结果显示,分别有54个上调基因和44个下调基因可被ame-miR-6001-3p靶向结合;上调基因富集在磷酸肌醇代谢、胰岛素信号通路、Hippo信号通路和谷胱甘肽代�【Objective】The whole transcriptome sequencing of Apis mellifera ligustica 7-and 10-day-old workers’ midguts(Am7 and Am10) was previously conducted. In this study, the differential expression profile and regulation network of genes were investigated to reveal the molecular mechanism underlying the midgut development.【Method】Gene expressions were calculated based on FPKM(fragments per kilobase of transcript per million mapped reads) algorithm, and differentially expressed genes(DEGs) were gained following the standard |log2 fold change|≥1 and P≤0.05. Target mRNAs of ame-miR-6001-3p were predicted utilizing TargetFinder. Annotations of all DEGs in GO and KEGG databases were performed using related software. In addition, DEGs enriched in 13 signaling pathways including AMPK, P13 K-Akt, Wnt, cAMP, FoxO, Hippo, m TOR, Jak-STAT, Toll-like receptor, TGF-beta, Notch, MAPK and NF-κB, as well as DEGs targeted by ame-miR-6001-3p were screened out, followed by visualization of enrichment networks and regulation networks with Cytoscape. Finally, Stem loop RT-PCR and RT-qPCR were used to verify the expression and differential expression pattern of ame-miR-6001-3p and DEGs in Am7 and Am10.【Result】A total of 1 038 DEGs were identified in Am7 vs Am10 comparison group, including 515 up-and 523 down-regulated genes, respectively. These DEGs were associated with cellular process, metabolic process and catalytic activity, and significantly enriched in some material and energy metabolisms such as oxidative phosphorylation, amino sugar and nucleotide sugar metabolisms, fatty acid metabolism and purine metabolism, indicative of the active cellular and metabolic activities. Expression cluster analysis suggested that 20, 18, 15 and 14 DEGs were respectively enriched in AMPK signaling pathway, PI3 K-Akt signaling pathway, endocytosis and Hippo signaling pathway. In addition, 57 DEGs were enriched in the aforementioned 13 signaling pathways associated with growth and development as well as immune defense, among them one DE

关 键 词：意大利蜜蜂 中肠 发育 差异表达基因 竞争性内源RNA 调控网络 

分 类 号：S891[农业科学—特种经济动物饲养]