中国农业科学 ›› 2018, Vol. 51 ›› Issue (21): 4197-4209.doi: 10.3864/j.issn.0578-1752.2018.21.018

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意大利蜜蜂幼虫肠道发育过程中的差异表达 microRNA及其调控网络

郭睿1(),杜宇1(),熊翠玲1,郑燕珍1,付中民1,徐国钧1,王海朋1,陈华枝1,耿四海1,周丁丁1,石彩云1,赵红霞2,陈大福1()   

  1. 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
  • 收稿日期:2018-05-22 接受日期:2018-06-28 出版日期:2018-11-01 发布日期:2018-11-01
  • 通讯作者: 郭睿,杜宇,陈大福 E-mail:ruiguo@fafu.edu.cn;m18505700830@163.com;dfchen826@fafu.edu.cn
  • 基金资助:
    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);福建省科技计划项目(2018J05042);福建省教育厅中青年教师教育科研项目(JAT170158);福建农林大学科技创新专项基金(CXZX2017343);福建农林大学科技发展基金(KF2015123)

Differentially Expressed MicroRNA and Their Regulation Networks During the Developmental Process of Apis mellifera ligustica Larval Gut

Rui GUO1(),Yu DU1(),CuiLing XIONG1,YanZhen ZHENG1,ZhongMin FU1,GuoJun XU1,HaiPeng WANG1,HuaZhi CHEN1,SiHai GENG1,DingDing ZHOU1,CaiYun SHI1,HongXia ZHAO2,DaFu CHEN1()   

  1. 1College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou 350002
    2Guangdong Institute of Applied Biological Resources, Guangzhou 510260
  • Received:2018-05-22 Accepted:2018-06-28 Online:2018-11-01 Published:2018-11-01
  • Contact: Rui GUO,Yu DU,DaFu CHEN E-mail:ruiguo@fafu.edu.cn;m18505700830@163.com;dfchen826@fafu.edu.cn

摘要:

【目的】微小RNA(microRNA,miRNA)是一类在转录后水平对mRNA进行负调控的关键调控因子。本研究旨在通过分析意大利蜜蜂(Apis mellifera ligustica,简称意蜂)幼虫肠道发育过程中差异表达miRNA(differentially expressed miRNA,DEmiRNA)及其调控网络,提供miRNA的表达谱和差异表达信息,揭示DEmiRNA在幼虫肠道发育中的作用。【方法】利用small RNA-seq(sRNA-seq)技术对意蜂4、5和6日龄幼虫肠道样品(Am4、Am5和Am6)进行测序,将质控后的数据与西方蜜蜂(Apis mellifera)参考基因组进行比对,然后将比对上的序列标签(tags)注释到miRBase数据库,并利用TPM(tags per million)算法归一化处理所得miRNA的表达量,再通过相关生物信息学软件对miRNA进行表达量聚类、前体二级结构预测及差异表达分析。利用TargetFinder软件预测DEmiRNA的靶基因,使用Blast软件对靶基因进行GO和KEGG数据库注释,进而通过Cytoscape软件构建miRNA-mRNA的调控网络。采用茎环实时荧光定量PCR(Stem-loop RT-qPCR)验证测序数据的可靠性。【结果】意蜂幼虫肠道样品的测序分别得到10 841 644、12 037 678和9 230 496条有效序列标签;Am4 vs Am5比较组包含16个上调和10个下调miRNA,Am5 vs Am6比较组包含5个上调和7个下调miRNA。其中,novel-m0031-3p为两个比较组所共有,并结合5个与蜕皮激素诱导蛋白相关的靶基因;二者的特有DEmiRNA数分别为25和11个。Am4 vs Am5的26个DEmiRNA结合5 742个靶基因,其中2 725个靶基因可注释到GO数据库中的46个GO term,并主要富集在结合、细胞进程、代谢进程和单组织进程等;Am5 vs Am6的12个DEmiRNA结合3 733个靶基因,且其中2 725个靶基因富集在结合、细胞进程、单组织进程和代谢进程等41个GO term;此外,两个比较组中的DEmiRNA分别有1 046和676个靶基因可注释到116和92条KEGG代谢通路,且Am4 vs Am5比较组的DEmiRNA的靶基因富集在Wnt信号通路、Hippo信号通路、嘌呤代谢和内吞作用等通路上的数量均高于Am5 vs Am6比较组。进一步分析结果显示Am4 vs Am5中的上调和下调miRNA可分别结合611和85个靶基因,其中ame-miR-6052结合的靶基因数最多,可通过结合5个靶基因,参与对细胞色素P450的调控;miR-281-x结合49个靶基因,并间接调控组氨酸代谢、TGF-β信号通路以及Hippo信号通路;Am5 vs Am6中的上调和下调miRNA可分别结合43和431个靶基因,其中miR-iab-4-x结合靶基因数量最多,并广泛参与调控背腹轴的形成、Hippo信号通路、Wnt信号通路、FoxO信号通路、Notch信号通路以及mTOR信号通路等与生长发育相关的通路。调控网络分析结果表明,DEmiRNA与靶基因间形成较为复杂的调控网络,DEmiRNA居于调控网络的中心位置,而mRNA位于调控网络的外周。最后,通过茎环实时荧光定量PCR对随机选取的3个DEmiRNA进行验证,结果证实了测序数据的可靠性。【结论】在全基因组水平对意蜂幼虫肠道的DEmiRNA及其靶基因进行预测和分析,并对DEmiRNA的调控网络进行构建及分析,发现意蜂幼虫通过调节包括ame-miR-6052、miR-iab-4-x、miR-281-x和novel-m0031-3p在内的多个miRNA的表达水平对肠道生长和发育进行调控,研究结果不仅提供了意蜂肠道发育过程的miRNA表达谱及差异表达信息,也为阐明意蜂幼虫肠道的发育机理打下基础。

关键词: 意大利蜜蜂, 幼虫肠道, 发育, 微小RNA, 靶基因, 调控网络

Abstract:

【Objective】MicroRNA (miRNA) is a kind of key regulator for negative regulation of mRNA at post-transcriptional level. The objective of this study is to provide miRNA expression patterns and differential expression information, illuminate the function of differentially expressed miRNA (DEmiRNA) in the development of larval gut by comprehensively investigating the DEmiRNAs and their regulation networks during the developmental process of Apis mellifera ligustica larval gut. 【Method】Deep sequencing of the 4-, 5- and 6-day-old larval guts of A. m. ligustica was conducted using small RNA-seq (sRNA-seq) technology, followed by mapping of the data after quality-control with the reference genome of Apis mellifera, and the mapped tags were then compared to miRBase database. The miRNA expression level was normalized by TPM algorithm, and the expression clustering, prediction of secondary structure of precursor and differential expression analysis were performed using related bioinformatic softwares. TargetFinder software was used to predict target gene of DEmiRNA, which was annotated to GO and KEGG databases using Blast, furthermore, miRNA-mRNA regulation networks were constructed using Cytoscape software. Stem-loop RT-qPCR was used to verify the sequencing data in this study.【Result】High-throughput sequencing of larval gut samples produced 10 841 644, 12 037 678 and 9 230 496 clean tags, respectively. In Am4 vs Am5 comparison group, there were16 up-regulated and 10 down-regulated miRNAs, while Am5 vs Am6 comparison group included 5 up-regulated and 7 down-regulated miRNAs, respectively. Among them, Novel-m0031-3p was shared by both Am4 vs Am5 and Am5 vs Am6, binding 5 target genes associated with ecdysone inducible protein, 25 and 11 DEmiRNAs were specific for the above-mentioned two comparison groups. DEmiRNA in Am4 vs Am5 could bind 5 742 target genes, among them 2 725 targets could be annotated to 46 GO terms in GO database, and the largest ones were binding, cellular process, metabolic process and single-organism process. Similarly, 12 DEmiRNAs in Am5 vs Am6 could link 3 733 target genes, among them 2 725 targets could be annotated to 41 GO terms, and mostly enriched terms were binding, cellular process, single-organism process and metabolic process. In addition, 1 046 and 676 target genes of two comparison groups were related to 116 and 92 KEGG pathways, and the number of DEmiRNA target genes in Am4 vs Am5 was more than that in Am5 vs Am6, which annotated to Wnt signaling pathway, Hippo signaling pathway, purine metabolism and endocytosis. Further analysis demonstrated that up-regulated and down-regulated miRNAs in Am4 vs Am5 could bind 611 and 85 target genes, and ame-miR-6052 linked the most target genes and participated in regulating cytochrome P450 via 5 target genes. miR-281-x could bind 49 target genes and indirectly regulate histidine metabolism, TGF-β signaling pathway and Hippo signaling pathway. In Am5 vs Am6 comparison group, up-regulated and down-regulated miRNAs could bind 43 and 431 target genes, respectively, among them miR-iab-4-x linked the most target genes, and it could participate in regulating growth and development related pathways, such as dorso-ventral axis formation, Hippo signaling pathway, Wnt signaling pathway, FoxO signaling pathway, Notch signaling pathway and mTOR signaling pathway. Regulation network analysis indicated that complex networks formed between DEmiRNAs and target genes, and DEmiRNAs lied in the center while target genes lied in the periphery. Finally, Stem-loop RT-qPCR was carried out to validate the randomly selected three DEmiRNAs, and the result confirmed the reliability of sequencing data. 【Conclusion】The DEmiRNA and corresponding target genes in the A. m. ligustica larval gut were predicted and analyzed at genome-wide level, it was found that A. m. ligustica are capable of regulating the expression of many miRNAs such as ame-miR-6052, miR-iab-4-x, miR-281-x and novel-m0031-3p. The results not only offer the expression pattern and differential expression information of miRNA during the developmental process of A. m. ligustica larval gut, but also lay a foundation for clarifying the molecular mechanisms underlying the larval gut’s development.

Key words: Apis mellifera ligustica, larval gut, development, microRNA, target gene, regulation network