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^,11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

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

GUO Rui^,1, DU Yu^,1, XIONG CuiLing¹, ZHENG YanZhen¹, FU ZhongMin¹, XU GuoJun¹, WANG HaiPeng¹, CHEN HuaZhi¹, GENG SiHai¹, ZHOU DingDing¹, SHI CaiYun¹, ZHAO HongXia², CHEN DaFu^,1 1College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou 350002
2Guangdong Institute of Applied Biological Resources, Guangzhou 510260

通讯作者: 郭睿,E-mail： ruiguo@fafu.edu.cn。 杜宇,E-mail： m18505700830@163.com。 陈大福,E-mail： dfchen826@fafu.edu.cn

第一联系人: 郭睿和杜宇为同等贡献作者。
责任编辑: 岳梅
收稿日期:2018-05-22接受日期:2018-06-28网络出版日期:2018-11-01

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

Received:2018-05-22Accepted:2018-06-28Online:2018-11-01


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.
Keywords：Apis mellifera ligustica;larval gut;development;microRNA;target gene;regulation network


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本文引用格式
郭睿, 杜宇, 熊翠玲, 郑燕珍, 付中民, 徐国钧, 王海朋, 陈华枝, 耿四海, 周丁丁, 石彩云, 赵红霞, 陈大福. 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[J]. 中国农业科学, 2018, 51(21): 4197-4209 doi:10.3864/j.issn.0578-1752.2018.21.018
GUO Rui, DU Yu, XIONG CuiLing, ZHENG YanZhen, FU ZhongMin, XU GuoJun, WANG HaiPeng, CHEN HuaZhi, GENG SiHai, ZHOU DingDing, SHI CaiYun, ZHAO HongXia, CHEN DaFu. Differentially Expressed MicroRNA and Their Regulation Networks During the Developmental Process of Apis mellifera ligustica Larval Gut[J]. Scientia Acricultura Sinica, 2018, 51(21): 4197-4209 doi:10.3864/j.issn.0578-1752.2018.21.018

0 引言

【研究意义】微小RNA（microRNA,miRNA）是一类长度约为18—25个核苷酸的非编码RNA（non-coding RNA,ncRNA）,通过对mRNA的负调控作用而广泛参与动物^[1,2]、植物^[3,4]和微生物^[5,6]的各类生物学过程。意大利蜜蜂（Apis mellifera ligustica,简称意蜂）是我国养蜂生产中的主要蜂种,在经济创收和生态维持等方面具有重要价值。利用small RNA-seq（sRNA-seq）技术对意蜂4、5和6日龄幼虫肠道进行测序,通过生物信息学分析方法对肠道发育过程中的差异表达miRNA（differentially expressed miRNA,DEmiRNA）进行全面分析,并分别构建上调和下调miRNA的调控网络,对揭示意蜂幼虫肠道发育过程中miRNA的差异表达规律、肠道发育相关关键miRNA的筛选和功能研究以及阐明肠道发育的机理具有重要意义。【前人研究进展】西方蜜蜂（Apis mellifera）基因组^[7]的公布为其组学和分子生物学研究奠定了基础。此后,随着高通量测序技术、生物信息学分析方法和软件的革新、融合及应用,蜜蜂的转录组学研究取得了较大进展^[8,9]。对蜜蜂的miRNA开展了一些研究,如ASHBY等^[10]研究发现,miR-184、miR-bantam和miR315参与了蜜蜂的细胞分化、组织结构重塑以及级型分化;陈晓^[11]对蜂王卵巢激活、产卵抑制和产卵恢复过程中的DEmiRNA进行靶基因预测和分析,发现多个DEmiRNA与卵巢激活和产卵密切相关;石元元^[12]研究发现,ame-bantam、ame-let-7和ame-miR-8参与了东方蜜蜂（Apis cerana）4日龄蜂王幼虫和工蜂幼虫的Wnt信号通路,并与雌性蜜蜂级型分化的分子机理密切相关。此外,蜜蜂肠道不仅是食物消化、营养吸收和利用的主要场所,也是抵御病原入侵的重要免疫器官。前人对蜜蜂肠道的研究主要集中在肠道菌群的群落结构和功能预测方面^[13,14],但有关蜜蜂及其幼虫肠道发育的研究极为滞后,肠道发育的分子机理仍不明确。笔者所在课题组前期已在mRNA组学水平对意蜂幼虫肠道进行了全面的转录组学研究,通过差异表达基因（DEG）和趋势分析揭示了意蜂幼虫肠道发育过程中的基因表达谱和差异表达规律^[15],意蜂幼虫肠道响应球囊菌（Ascosphaera apis）胁迫的免疫应答^[16],以及意蜂幼虫与球囊菌之间的互作^[17]。【本研究切入点】目前为止,意蜂幼虫肠道发育过程中的miRNA表达谱仍然缺失,相关miRNA在幼虫肠道发育过程中的作用还不明确。【拟解决的关键问题】结合sRNA-seq技术对意蜂4、5和6日龄幼虫肠道样品进行测序和分析,通过DEmiRNA及其靶基因的预测和分析、调控网络的构建和分析,提供意蜂幼虫肠道发育过程中的miRNA表达谱和差异表达信息,揭示DEmiRNA在肠道发育中的作用。

1 材料与方法

试验于2017年9月至2018年5月在福建农林大学蜂学学院蜜蜂保护实验室进行。

1.1 生物材料

供试意蜂幼虫取自福建农林大学蜂学学院教学蜂场。

1.2 意蜂幼虫人工饲养及样品测序

按照王倩等^[18]的方法配制幼虫饲料,从健康意蜂蜂群中提取巢脾,将2日龄幼虫移至已预置50 μL饲料的24孔细胞培养板中,在35℃,相对湿度（RH）为90%的培养箱中饲养,每隔24 h更换饲料。分别剖取4、5和6日龄意蜂幼虫肠道（分别记为Am4、Am5和Am6）,装入EP管后放入液氮速冻,随后保存在-80℃的超低温冰箱。进行3次生物学重复。Am4的3个生物学重复分别为Am4-1、Am4-2和Am4-3,Am5的3个生物学重复分别为Am5-1、Am5-2和Am5-3,Am6的3个生物学重复分别为Am6-1、Am6-2和Am6-3。上述9个肠道样品委托广州基迪奥生物科技有限公司对进行单端测序,测序平台为Illumina MiSeq。原始数据已上传NCBI SRA数据库,BioProject号：PRJNA408312。

1.3 测序数据的质控及定位

对原始下机数据,利用Perl脚本剔除衔接子（adaptor）、未知碱基N和低质量的reads,从而得到高质量的序列（clean reads）,保证后续数据分析的准确性。通过Bowite软件将各样品的tags序列比对到GenBank及Rfam（11.0）数据库,过滤核糖体RNA（rRNA）、胞质小RNA（scRNA）以及核内小RNA（snoRNA）等ncRNA和重复序列,得到miRNA的非注释标签序列（unannotated tags）。继而利用Bowit 软件将非注释标签序列与西方蜜蜂的参考基因组（assembly Amel 4.5）的序列进行比对,得到相关tags在参考基因组上的位置信息,即为mapped tags。

1.4 DEmiRNA的预测

利用miRDeep2软件^[19]将mapped tags与miRBase数据库中已知miRNA前体序列进行比对,从而鉴定已知miRNA的表达,同时得到可能的前体序列。对各样本中miRNA进行表达量的统计,并通过TPM（tags per million）算法公式（TPM=T×10⁶/N,T表示miRNA的tags,N表示总miRNA的tags）对全部miRNA的表达量进行归一化处理。利用R软件计算各样品之间的相关性系数。DEmiRNA（Am4 vs Am5、Am5 vs Am6和Am4 vs Am6）的筛选标准为P-value≤0.05且|log₂ Fold change|≥1。

1.5 DEmiRNA靶基因预测及分析

根据DEmiRNA与对应物种的基因序列信息,利用TargetFinder软件^[20]进行靶基因预测,并利用Blast软件将预测靶基因序列与GO（Gene Ontology）、KEGG数据库比对,获得靶基因的注释信息。利用RNAhybrid、TargetScan和Miranda软件预测DEmiRNA结合的靶基因,根据上述靶向结合关系构建miRNA-mRNA的调控网络并通过Cytoscape软件^[21]将其可视化。

1.6 DEmiRNA的茎环实时荧光定量PCR（Stem-loop RT-qPCR）验证

为了验证sRNA-seq数据的可靠性,随机选取3个DEmiRNA（miR-7964-y、miR-8516-x和miR-3747-x）进行Stem-loop RT-qPCR验证。根据所选DEmiRNA的序列,参照CHEN等^[22]的方法,利用DNAMAN软件（Lynnon Biosoft公司,美国）设计特异性的Stem-loop引物、上游引物和下游引物,委托上海生工生物工程有限公司进行引物合成。选择snRNA U6作为内参。利用RNA抽提试剂盒（Axygen公司,美国）分别提取Am4、Am5和Am6的总RNA,利用Stem-loop引物进行反转录得到相应的cDNA,作为模板进行qPCR。反应体系（20 μL）中含有SYBR Green Dye 10 μL,上下游引物各1 μL,cDNA模板DNA 1 μL,Rox 0.44 μL,DEPC水补至20 μL。在ABI 7500荧光定量PCR仪（ABI公司,美国）中进行反应,反应条件：95℃预变性1 min,95℃变性15 s,48℃延伸30 s,共40个循环,最后72℃延伸45 s。所选miRNA的相对表达量采用2^-△△Ct法计算。每个反应进行3个生物学重复和3次平行重复。

2 结果

2.1 数据质控与评估

本研究中,3个幼虫肠道样品的sRNA-seq分别产生13 186 921、14 255 967和10 921 897条clean reads,经严格过滤后得到的clean tags数分别为10 841 644（82.22%）、12 037 678（84.44%）和9 230 496（84.51%）条（表1）。Am4、Am5和Am6组内各生物学重复之间的Pearson相关系数均在0.9734以上,说明各样品的重复性较好（图1）。上述结果说明本研究的测序数据质量良好,可用于进一步分析。

Table 1
表1
表1sRNA-seq数据统计
Table 1Overview of sRNA-seq datasets

样品Sample	有效读段Clean reads	有效标签序列Clean tags
Am4-1	14736731	11802658 (80.09%)
Am4-2	12954535	10940303 (84.45%)
Am4-3	11869496	9781972 (82.41%)
Am5-1	13907726	11653319 (83.79%)
Am5-2	13844605	11812820 (85.32%)
Am5-3	15015570	12646896 (84.23%)
Am6-1	11260874	9324373 (82.80%)
Am6-2	10434013	9038930 (86.63%)
Am6-3	11070804	9328185 (84.26%)

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图1

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图1各意蜂幼虫肠道样品的不同生物学重复间的Pearson相关性

Fig. 1Pearson correlation between different biological repeats within each A. m. ligustica larval gut sample

2.2 意蜂幼虫肠道的DEmiRNA分析

Am4 vs Am5比较组中有26个miRNA差异表达,包括16个上调miRNA和10个下调miRNA。Am5 vs Am6 比较组共有12个DEmiRNA,包括5个上调miRNA和7个下调miRNA。Am 4 vs Am6比较组中有41个miRNA差异表达,包括15个上调miRNA和26个下调miRNA。分别对Am4 vs Am5和Am5 vs Am6的DEmiRNA进行表达量聚类分析,结果显示2个比较组中DEmiRNA的变化倍数差异明显,多数DEmiRNA的差异表达幅度较小（图2-A、2-B）。进一步分析发现,Am4 vs Am5和Am5 vs Am6中DEmiRNA分别包含3个和2个novel miRNA,其中novel-m0031-3p为2个比较组所共有。利用miRDeep2软件对novel miRNA的前体二级结构进行预测,结果显示它们均有标志性的茎环结构（图2-C—2-F）。

图2

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图2DEmiRNA的表达量聚类及novel miRNA前体的二级结构

A：Am4 vs Am5中DEmiRNA的表达量聚类Expression clustering of DEmiRNA in Am4 vs Am5;B：Am5 vs Am6中DEmiRNA的表达量聚类Expression clustering of DEmiRNA in Am5 vs Am6;C：novel-m0004-3p前体的二级结构Secondary structure of precursor of novel-m0004-3p;D：novel-m0017-3p前体的二级结构Secondary structure of precursor of novel-m0017-3p;E：novel-m0031-3p前体的二级结构Secondary structure of precursor of novel-m0031-3p;F：novel-m0037-5p前体的二级结构Secondary structure of precursor of novel-m0037-5p。黄色区域为novel miRNA的成熟序列Yellow regions indicate mature sequences of novel miRNA
Fig. 2Expression clustering of DEmiRNA and secondary structure of novel miRNA’ precursors

2.3 意蜂幼虫肠道的DEmiRNA的靶基因预测及功能注释

利用TargetFinder软件对意蜂幼虫肠道的DEmiRNA进行靶基因预测,Am4 vs Am5、Am5 vs Am6的DEmiRNA可分别预测出5 742和3 733个靶基因。对上述靶基因进行GO数据库注释,结果显示Am4 vs Am5的DEmiRNA的2 725个靶基因共涉及46个GO term,富集基因数最多的是结合（1 612 gene）、细胞进程（1 537 gene）、代谢进程（1 232 gene）、单组织进程（1 197 gene）、催化活性（1 020 gene）、细胞膜（761 gene）、细胞（757 gene）、细胞组件（757 gene）、细胞膜组件（745 gene）、细胞器（578 gene）等（图3）;Am5 vs Am6的DEmiRNA的1 785个靶基因可注释到41个GO term,富集基因数最多的是结合（1 068 gene）、细胞进程（1 026 gene）、单组织进程（847 gene）、代谢进程（799 gene）、催化活性（591 gene）、细胞膜（533 gene）、细胞膜组件（529 gene）、细胞（448 gene）、细胞组件（448 gene）、生物学调控（348 gene）等（图3）。说明随着发育时间的延长,意蜂幼虫肠道的DEmiRNA数、靶基因数及其涉及的GO term数逐渐减少;DEmiRNA广泛参与意蜂幼虫肠道的新陈代谢、细胞生命活动及免疫防御。

图3

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图3意蜂幼虫肠道DEmiRNA的靶基因的GO数据库注释

Fig. 3GO database annotation of DEmiRNA target genes in the larval guts of A. m. ligustica



进一步对DEmiRNA的靶基因进行KEGG代谢通路（pathway）富集分析,结果显示Am4 vs Am5中的1 046个DEmiRNA的靶基因可注释到116条pathway,其中富集基因数最多的是Wnt信号通路（140 gene）、嘌呤代谢（87 gene）、Hippo信号通路（80 gene）、内吞作用（64 gene）、光传导（60 gene）、神经活性配体-受体相互作用（57 gene）、FoxO信号通路（43 gene）、内质网中蛋白质的加工（41 gene）、磷脂酰肌醇信号系统（38 gene）、泛素介导的蛋白水解（36 gene）等（图4-A）,说明相应的miRNA参与到意蜂4和5日龄幼虫肠道发育过程中的新陈代谢、蛋白质合成、免疫防御以及相关信号通路的调控。

图4

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图4意蜂幼虫肠道DEmiRNA的靶基因的KEGG数据库注释

A：Am4 vs Am5中的DEmiRNA的靶基因Target genes of DEmiRNA in Am4 vs Am5;B：Am5 vs Am6中的DEmiRNA的靶基因Target genes of DEmiRNA in Am5 vs Am6
Fig. 4KEGG database annotation of DEmiRNA target genes in the larval guts of A. m. ligustica



Am5 vs Am6中的676个DEmiRNA的靶基注释到92条pathway,并主要富集在Wnt信号通路（109 gene）、光传导（69 gene）、Hippo信号通路（66 gene）、神经活性配体-受体相互作用（57 gene）、嘌呤代谢（42 gene）、内吞作用（38 gene）、背腹轴形成（30 gene）、mRNA监视（28 gene）、Hedgehog信号通路、昼夜节律（24 gene）等（图4-B）,说明相应的miRNA同样广泛参与了意蜂幼虫5和6日龄肠道的生长发育过程中各类新陈代谢以及信号通路的调控过程。

2.4 意蜂幼虫肠道DEmiRNA的调控网络分析

利用软件预测DEmiRNA结合的靶基因并通过Cytoscape软件进行可视化,结果显示Am4 vs Am5中上调miRNA可结合611个靶基因,下调miRNA可结合85个靶基因,上调（或下调）miRNA与靶基因形成较为复杂的调控网络,DEmiRNA居于调控网络的中心位置,而靶基因处于调控网络的外周;其中,所有DEmiRNA均可连接2个靶基因以上,ame-miR- 6052结合的靶基因数多达204个（图5-A、5-B）。Am5 vs Am6中上调和下调miRNA可分别结合43和431个靶基因,上调（或下调）miRNA同样与靶基因形成复杂的调控网络,所有DEmiRNA均可连接3个以上的靶基因,其中miR-iab-4-x结合的靶基因数最多,达到125个（图5-C、5-D）。

图5

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图5意蜂幼虫肠道发育过程中DEmiRNA的调控网络

A：Am4 vs Am5中上调miRNA的miRNA-mRNA网络miRNA-mRNA networks of up-regulated miRNA in Am4 vs Am5;B：Am4 vs Am5中下调miRNA的miRNA-mRNA网络miRNA-mRNA networks of down-regulated miRNA in Am4 vs Am5;C：Am5 vs Am6中上调miRNA的miRNA-mRNA网络miRNA-mRNA networks of up-regulated miRNA in Am5 vs Am6;D：Am5 vs Am6中下调miRNA的miRNA-mRNA网络miRNA-mRNA networks of down-regulated miRNA in Am5 vs Am6
Fig. 5Regulation networks of DEmiRNA during the developmental process of A. m. ligustica larval gut

2.5 意蜂幼虫肠道DEmiRNA的RT-qPCR验证

随机挑取3个DEmiRNA（miR-7964-y、miR-8516-x和miR-3747-x）进行RT-qPCR验证,结果显示它们的表达水平的变化趋势和测序数据中相应DEmiRNA的表达水平的变化趋势一致（图6）,说明本研究中的测序数据真实可靠。

图6

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图6DEmiRNA的RT-qPCR验证

Fig. 6RT-qPCR validation of DEmiRNA

A：miR-7964-y;B：miR-8516-x;C：miR-3747-x

3 讨论

MiRNA作为一种重要的基因表达调控因子,在昆虫的各种生物学进程发挥关键作用^[23]。家蚕（Bombyx mori）和小菜蛾（Plutella xylostella）不同发育阶段和不同组织中的miRNA表达谱研究揭示了miRNA在其生长发育^[24]、变态^[25]及行为反应^[26]等过程中的重要调控功能。相比于果蝇、家蚕等模式昆虫,蜜蜂的miRNA研究相对滞后。陈璇等^[27,28]曾对三型蜂不同发育阶段的混合小RNA文库进行了测序,发现了267个novel miRNA,并进一步对蜂王和工蜂4和5日龄幼虫的DEmiRNA及其靶基因进行了分析,为蜜蜂发育和级型分化关键时期的miRNA调控网络研究打下了基础。目前,有关蜜蜂幼虫肠道发育过程miRNA表达谱的研究十分滞后,miRNA在肠道发育中的调控机理仍不明确。本研究利用sRNA-seq技术对意蜂4、5和6日龄幼虫肠道进行测序,通过生物信息学方法对邻近日龄幼虫肠道的miRNA进行差异表达分析,分别预测出26和12个DEmiRNA,说明DEmiRNA的数量在意蜂幼虫肠道发育过程中有逐渐减少的趋势;进一步分析发现Am4 vs Am5和Am5 vs Am6中的特有DEmiRNA数分别为25（miR-342-y、ame-miR-3759和miR-4331-y等）和11个（miR-4955-x、miR-46-y和ame-miR-3478等）,二者包含1个共有DEmiRNA（novel-m0031-3p）,且novel-m0031-3p的表达水平随日龄的增加呈逐渐下调趋势,推测其通过下调表达量减少对靶基因的抑制作用,从而在意蜂幼虫肠道的不同发育阶段均发挥基础性的调控作用,而特有DEmiRNA在幼虫肠道的不同发育阶段发挥特殊的调控功能。

蜜蜂幼虫肠道内存在简单的共生菌,为尽量减小意蜂幼虫肠道内共生菌对测序数据的影响,本研究一方面在人工饲喂过程中尽量减少环境微生物的带入,例如对所用器具进行高压蒸汽灭菌,用75%酒精对人工气候箱进行擦洗消毒,更换饲料时在酒精灯旁操作等;另一方面通过对原始数据进行严格的过滤和质控、比对西方蜜蜂基因组以消除肠道共生菌数据的影响,昆虫和微生物的物种亲缘关系较远,二者的基因保守性很低,因而比对西方蜜蜂基因组的数据理论上应为意蜂幼虫肠道本身的数据。

蜜蜂肠道不仅是食物消化、营养吸收和利用的主要场所,也是抵御病原入侵的重要免疫器官。本研究发现,Am5 vs Am6中的12个DEmiRNA的靶基因富集在了各类能量和物质代谢通路,如氮代谢（1 gene）、氨基酸代谢（32 gene）、碳水化合物代谢（69 gene）和脂质代谢（74 gene）等,表明相应的DEmiRNA参与了意蜂幼虫肠道能量和物质代谢的调控;还发现部分靶基因注释到与生长、发育相关的代谢通路和信号通路,包括背腹轴形成（30 gene）、Hippo信号通路（66 gene）、Wnt信号通路（109 gene）等,表明相应的DEmiRNA在生长和发育过程中发挥重要的调控作用。此外,分别有38和13个靶基因注释到内吞作用和泛素介导的蛋白质水解等细胞免疫通路,分别有6和4个靶基因注释到MAPK和Jak-STAT等体液免疫通路,表明相应的DEmiRNA参与了意蜂幼虫肠道的免疫防御过程。本研究中,Am4 vs Am5中的26个DEmiRNA的靶基因同样也广泛参与生长发育、新陈代谢以及免疫防御相关的代谢通路。Am4 vs Am5中的DEmiRNA及其靶基因数均高于Am5 vs Am6,意蜂5日龄幼虫体积近乎4日龄幼虫的2倍,但6日龄幼虫体积稍大于5日龄幼虫,推测意蜂4—5日龄幼虫的发育时期需要更多的DEmiRNA参与到生长发育、新陈代谢等方面的调控。

蜕皮激素（ecdysteroid,Ec）是昆虫体内的重要激素之一,参与昆虫生长、变态和生殖的整个生命活动,其滴度的阶段性增加是昆虫生长和发育的必要条件^[29]。novel-m0031-3p在Am4 vs Am5和Am5 vs Am6中均有表达,且其结合的5个靶基因（XM_006564318.2、XM_006564319.2、XM_006564320.2、XM_006564321.2和XM_016914663.1）均涉及Ec诱导蛋白的调控,有研究表明蜕皮触发激素基因可在家蚕幼虫蜕皮和变态发育过程中起到关键调控作用^[30],因此推测novel-m0031-3p通过调控Ec滴度使其达到动态平衡,从而保证意蜂幼虫肠道的正常发育。miR-281是一种高度保守的miRNA,在家蚕幼虫阶段中呈高量表达,与神经发育、组织生长密切相关^[25]。周艳河^[31]研究发现,受到高剂量登革病毒侵染的白纹伊蚊（Aedes albopictus）中肠内miR-281会靶向调控自身基因从而影响病毒的复制水平;熊慧萍^[32]对位于可变内含子区的黑腹果蝇（Drosophila melanogaster）的miR-281-1/2基因转录和启动子进行分析,发现miR-281只在3龄幼虫、蛹和成虫期表达,且在不同发育阶段miR-281转录起始位点的转录效率存在差异。本研究中,miR-281-x是Am4 vs Am5中的特有DEmiRNA,其结合的49个靶基因涉及TGF-β信号通路（8 gene）、Hippo信号通路（7 gene）、背腹轴的形成（3 gene）和组氨酸代谢（3 gene）等代谢通路,推测其参与对意蜂幼虫肠道早期生长和发育的调控,但miR-281-x的时空表达谱需进一步研究。miR-iab-4可通过调控Hox编码蛋白的Ubx基因表达间接影响黑腹果蝇的翅形成过程,miR-iab-4的突变会影响果蝇幼虫的自身调节能力^[33]。本研究中,miR-iab-4-x为Am5 vs Am6中的特有DEmiRNA,其结合的125个靶基因涉及Hippo信号通路（14 gene）、Wnt信号通路（9 gene）、FoxO信号通路（8 gene）、Notch信号通路（7 gene）、mTOR信号通路（6 gene）和背腹轴的形成（5 gene）等与生长发育相关的代谢通路,推测其参与意蜂幼虫肠道发育后期的调控,miR-iab-4-x的下调表达可能对意蜂幼虫肠道发育过程发挥重要作用。

一个miRNA可以同时靶向调控多个mRNA,反之亦然。为进一步揭示DEmiRNA的作用,本研究通过序列匹配关系预测DEmiRNA结合的靶基因,并构建了二者的调控网络（图5）,发现部分上调和下调的miRNA位于调控网络的中心位置且结合较多的mRNA,如ame-miR-6052与miR-iab-4-x可分别结合204和125个靶基因,具有很高的连通性,表明二者可能在意蜂幼虫肠道的生长和发育过程中发挥关键的调控功能。细胞色素P450作为一种重要的解毒酶,广泛存在于昆虫的脂肪体、马氏管和中肠内,且在中肠的含量最高;其也可参与内源性物质代谢,在生物体内发挥重要的作用^[34,35]。本研究发现Am4 vs Am5中特有的ame-miR-6052表达量上调,其结合的5个靶基因（XM_006559340.2、XM_006559341.1、XM_016912202.1、XM_016916903.1和XM_623618.5）与细胞色素P450的调节有关,推测ame-miR-6052可参与意蜂幼虫肠道对异源物质的降解、新陈代谢过程的调控,下一步可通过合成miRNA mimic、miRNA inhibitor对其进行过表达和敲减,从而深入探究其功能。

4 结论

结合高通量测序技术和生物信息学分析方法对意蜂幼虫肠道的DEmiRNA进行了全基因组水平的预测和分析,并对DEmiRNA-mRNA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了基础。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。

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蜜蜂的级型分化被证实是由蜂王浆中的Royalactin决定,工蜂和蜂王幼虫在级型分化时编码基因的表达差异也被广泛研究.我们发现,在蜜蜂幼虫的级型分化过程中,lncRNA也有着显著的表达差异,因此认为,lncRNA也参与了蜜蜂的级型分化过程.进一步的分析显示,lncRNA可能通过影响上下游基因的转录和功能执行的方式,在蜜蜂早期发育的多细胞组织发育、神经系统发育和转录调控的过程中起到重要的调控作用.

[10] ASHBY R, FORêT S, SEARLE I, MALESZKA R . MicroRNAs in honey bee caste determination
Scientific Reports, 2016,6:18794.
DOI:10.1038/srep18794URLPMID:4704047 [本文引用: 1]
The cellular mechanisms employed by some organisms to produce contrasting morphological and reproductive phenotypes from the same genome remains one of the key unresolved issues in biology. Honeybees (Apis mellifera) use differential feeding and a haplodiploid sex determination system to generate three distinct organismal outcomes from the same genome. Here we investigate the honeybee female and male caste-specific microRNA and transcriptomic molecular signatures during a critical time of larval development. Both previously undetected and novel miRNAs have been discovered, expanding the inventory of these genomic regulators in invertebrates. We show significant differences in the microRNA and transcriptional profiles of diploid females relative to haploid drone males as well as between reproductively distinct females (queens and workers). Queens and drones show gene enrichment in physio-metabolic pathways, whereas workers show enrichment in processes associated with neuronal development, cell signalling and caste biased structural differences. Interestingly, predicted miRNA targets are primarily associated with non-physio-metabolic genes, especially neuronal targets, suggesting a mechanistic disjunction from DNA methylation that regulates physio-metabolic processes. Accordingly, miRNA targets are under-represented in methylated genes. Our data show how a common set of genetic elements are differentially harnessed by an organism, which may provide the remarkable level of developmental flexibility required.

[11] 陈晓 . 蜜蜂卵巢激活和产卵过程差异表达的编码RNA与非编码RNA的筛选和鉴定
[D]. 北京: 中国农业科学院, 2017.
[本文引用: 1]

CHEN X . Identification of differentially expressed coding and noncoding RNAs during ovary activation and oviposition in honey bees
[D]. Beijing: Chinese Academy of Agricultural Sciences, 2017. ( in Chinese)
[本文引用: 1]

[12] 石元元 . 东方蜜蜂遗传图谱构建以及雌性蜜蜂发育分子机理
[D]. 南昌: 江西农业大学, 2014.
[本文引用: 1]

SHI Y Y . Construction of genetic linkage map in Apis cerana and molecular mechanism of development in females honey bee
[D]. Nanchang: Jiangxi Agricultural University, 2014. ( in Chinese)
[本文引用: 1]

[13] 常伟 . 蜜蜂消化道共生细菌及其多态性研究初探
[D]. 福州: 福建农林大学, 2010.
[本文引用: 1]

CHANG W . Diversity of symbiotic bacteria in honeybee alimentary tract
[D]. Fuzhou: Fujian Agriculture and Forestry University, 2010. ( in Chinese)
[本文引用: 1]

[14] 贾慧茹 . 亚致死剂量吡虫啉对意大利蜜蜂中肠菌群的影响
[D]. 北京: 中国农业科学院, 2015.
[本文引用: 1]

JIA H R . Effect of the sublethal doses of imidacloprid on the bacterial diversity in the midgut of Apis mellifera ligustica
[D]. Beijing: Chinese Academy of Agricultural Sciences, 2015. ( in Chinese)
[本文引用: 1]

[15] 郭睿, 解彦玲, 熊翠玲, 尹伟轩, 郑燕珍, 付中民, 陈大福 . 意大利蜜蜂4、5和6日龄幼虫肠道发育过程中差异表达基因的趋势分析
上海交通大学学报(农业科学版), 2018,36(4):14-21, 29.
[本文引用: 1]

GUO R, XIE Y L, XIONG C L, YI W X, ZHENG Y Z, FU Z M, CHEN D F . Trend analysis for differentially expressed genes in developmental process of 4-, 5- and 6-day-old larval guts of Apis mellifera ligustica.
Journal of Shanghai Jiaotong University (Agricultural Science), 2018,36(4):14-21, 29. (in Chinese)
[本文引用: 1]

[16] CHEN D, GUO R, XU X, XIONG C L, LIANG Q, ZHENG Y Z, LUO Q, ZHANG Z, HUANG Z J, KUMAR D, XI W J, ZOU X, LIU M . Uncovering the immune responses of Apis mellifera ligustica, larval gut to Ascosphaera apis, infection utilizing transcriptome sequencing.
Gene, 2017,621:40-50.
[本文引用: 1]

[17] 陈大福, 郭睿, 熊翠玲, 梁勤, 郑燕珍, 徐细建, 黄枳腱, 张曌楠, 张璐, 李汶东, 童新宇, 席伟军 . 胁迫意大利蜜蜂幼虫肠道的球囊菌的转录组分析
昆虫学报, 2017,60(4):401-411.
DOI:10.16380/j.kcxb.2017.04.005URL [本文引用: 1]
【目的】本研究旨在通过趋势分析对胁迫意大利蜜蜂Apis mellifera ligustica（简称意蜂）幼虫肠道的球囊菌Ascosphaera apis的差异表达基因（DEGs）进行转录组分析。【方法】将纯化的球囊菌孢子配制为1×107孢子/m L的饲料饲喂意蜂3日龄幼虫,利用Illumina Hi Seq 2500平台对球囊菌胁迫的意蜂幼虫肠道c DNA进行测序,将过滤得到的有效读段（clean reads）映射（mapping）到核糖体数据库及意蜂参考基因组,最后将未映射上的reads映射到本课题组组装并注释的球囊菌参考转录组。利用STEM软件对DEGs进行趋势分析。利用WEGO软件对显著表达模式中的DEGs进行GO富集分析。利用Blastall对显著表达模式中的DEGs进行KEGG代谢通路富集分析。最后,通过对随机选取的6个DEGs进行RT-q PCR分析,对RNA-seq数据进行验证。【结果】球囊菌胁迫意蜂幼虫肠道样品的Illumina测序共得到球囊菌的25 454 076条原始读段（raw reads）,经过滤得到24 909 820条clean reads,Q30均在93.46%以上。趋势分析结果显示,19 893个DEGs聚类为8个表达模式,其中有12 151个DEGs聚类为3个表现为显著上调趋势的表达模式。GO富集分析结果显示,表现上调趋势的DEGs富集在40个GO term,富集基因数最多的为细胞进程（cellular process）（2 601 unigenes）,其次为代谢进程（metabolic process）（2 553 unigenes）和细胞（cell）（2 522unigenes）。KEGG代谢通路富集分析结果显示,上调趋势中的DEGs富集在119个代谢通路上,其中富集基因数最多的是核糖体（ribosome）（213 unigenes）,其次为氨基酸生物合成（biosynthesis of amino acids）（154 unigenes）和内质网蛋白加工（protein processing in endoplasmic reticulum）（130unigenes）。共有48个DEGs富集在MAPK信号通路上,聚类分析结果显示,这些DEGs随着胁迫时间的延长表达水平逐渐增强。RT-q PCR结果显示,6个DEGs的表达水平变化趋势与RNA-seq数据一致,证明
CHEN D F, GUO R, XIONG C L, LIANG Q, ZHENG Y Z, XU X J, HUANG Z J, ZHANG Z N, ZHANG L, LI W D, TONG X Y, XI W J . Transcriptomic analysis of Ascosphaera apis stressing larval gut of Apis mellifera ligustica(Hyemenoptera: Apidae).
Acta Entomologica Sinica, 2017,60(4):401-411. (in Chinese)
DOI:10.16380/j.kcxb.2017.04.005URL [本文引用: 1]
【目的】本研究旨在通过趋势分析对胁迫意大利蜜蜂Apis mellifera ligustica（简称意蜂）幼虫肠道的球囊菌Ascosphaera apis的差异表达基因（DEGs）进行转录组分析。【方法】将纯化的球囊菌孢子配制为1×107孢子/m L的饲料饲喂意蜂3日龄幼虫,利用Illumina Hi Seq 2500平台对球囊菌胁迫的意蜂幼虫肠道c DNA进行测序,将过滤得到的有效读段（clean reads）映射（mapping）到核糖体数据库及意蜂参考基因组,最后将未映射上的reads映射到本课题组组装并注释的球囊菌参考转录组。利用STEM软件对DEGs进行趋势分析。利用WEGO软件对显著表达模式中的DEGs进行GO富集分析。利用Blastall对显著表达模式中的DEGs进行KEGG代谢通路富集分析。最后,通过对随机选取的6个DEGs进行RT-q PCR分析,对RNA-seq数据进行验证。【结果】球囊菌胁迫意蜂幼虫肠道样品的Illumina测序共得到球囊菌的25 454 076条原始读段（raw reads）,经过滤得到24 909 820条clean reads,Q30均在93.46%以上。趋势分析结果显示,19 893个DEGs聚类为8个表达模式,其中有12 151个DEGs聚类为3个表现为显著上调趋势的表达模式。GO富集分析结果显示,表现上调趋势的DEGs富集在40个GO term,富集基因数最多的为细胞进程（cellular process）（2 601 unigenes）,其次为代谢进程（metabolic process）（2 553 unigenes）和细胞（cell）（2 522unigenes）。KEGG代谢通路富集分析结果显示,上调趋势中的DEGs富集在119个代谢通路上,其中富集基因数最多的是核糖体（ribosome）（213 unigenes）,其次为氨基酸生物合成（biosynthesis of amino acids）（154 unigenes）和内质网蛋白加工（protein processing in endoplasmic reticulum）（130unigenes）。共有48个DEGs富集在MAPK信号通路上,聚类分析结果显示,这些DEGs随着胁迫时间的延长表达水平逐渐增强。RT-q PCR结果显示,6个DEGs的表达水平变化趋势与RNA-seq数据一致,证明

[18] 王倩, 孙亮先, 肖培新, 刘锋, 康明江, 胥保华 . 室内人工培育中华蜜蜂幼虫技术研究
山东农业科学, 2009(11):113-116.
DOI:10.3969/j.issn.1001-4942.2009.11.037URL [本文引用: 1]
以中华蜜蜂（Apis cerana cerana,简称中蜂）幼虫为试验材料,研究了不同饲粮配方、饲养模式和培养板对中蜂幼虫发育的影响。得出以下结果：幼虫饲粮LD1和LD3对幼虫的饲喂效果显著优于LD2;比较不同移虫模式下幼虫发育成功率：日转移法为61.1%,不转移法为70.8%;其中,在不转移饲喂模式下,使用新培养板培养的发育成功率（69.8%）显著高于重复利用的培养板培养的发育成功率（39.6%）（P〈0.01）。表明室内以LD1或LD3饲喂,采用＂不转移法＂模式,并使用新培养板,更利于中蜂幼虫的发育。
WANG Q, SUN L X, XIAO P X, LIU F, KANG M J, XU B H . Study on technology for indoor artificial feeding ofApis cerana cerana larvae
Shandong Agricultural Sciences, 2009(11):113-116. (in Chinese)
DOI:10.3969/j.issn.1001-4942.2009.11.037URL [本文引用: 1]
以中华蜜蜂（Apis cerana cerana,简称中蜂）幼虫为试验材料,研究了不同饲粮配方、饲养模式和培养板对中蜂幼虫发育的影响。得出以下结果：幼虫饲粮LD1和LD3对幼虫的饲喂效果显著优于LD2;比较不同移虫模式下幼虫发育成功率：日转移法为61.1%,不转移法为70.8%;其中,在不转移饲喂模式下,使用新培养板培养的发育成功率（69.8%）显著高于重复利用的培养板培养的发育成功率（39.6%）（P〈0.01）。表明室内以LD1或LD3饲喂,采用＂不转移法＂模式,并使用新培养板,更利于中蜂幼虫的发育。

[19] FRIEDLäNDER M R, MACKOWIAK S D, LI N, CHEN W, RAJEWSKY N . MiRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades
Nucleic Acids Research, 2012,40(1):37-52.
DOI:10.1093/nar/gkr688URLPMID:21911355 [本文引用: 1]
Abstract microRNAs (miRNAs) are a large class of small non-coding RNAs which post-transcriptionally regulate the expression of a large fraction of all animal genes and are important in a wide range of biological processes. Recent advances in high-throughput sequencing allow miRNA detection at unprecedented sensitivity, but the computational task of accurately identifying the miRNAs in the background of sequenced RNAs remains challenging. For this purpose, we have designed miRDeep2, a substantially improved algorithm which identifies canonical and non-canonical miRNAs such as those derived from transposable elements and informs on high-confidence candidates that are detected in multiple independent samples. Analyzing data from seven animal species representing the major animal clades, miRDeep2 identified miRNAs with an accuracy of 98.6-99.9% and reported hundreds of novel miRNAs. To test the accuracy of miRDeep2, we knocked down the miRNA biogenesis pathway in a human cell line and sequenced small RNAs before and after. The vast majority of the >100 novel miRNAs expressed in this cell line were indeed specifically downregulated, validating most miRDeep2 predictions. Last, a new miRNA expression profiling routine, low time and memory usage and user-friendly interactive graphic output can make miRDeep2 useful to a wide range of researchers.

[20] ALLEN E, XIE Z, GUSTAFSON A M, CARRINGTON J C . MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants
Cell, 2005,121(2):207-221.
DOI:10.1016/j.cell.2005.04.004URLPMID:15851028 [本文引用: 1]
Plants and animals use small RNAs (microRNAs [miRNAs] and siRNAs) as guides for posttranscriptional and epigenetic regulation. In plants, miRNAs and trans-acting (ta) siRNAs form through distinct biogenesis pathways, although they both interact with target transcripts and guide cleavage. An integrated approach to identify targets of Arabidopsis thaliana miRNAs and ta-siRNAs revealed several new classes of small RNA-regulated genes, including conventional genes such as Argonaute2 and an E2-ubiquitin conjugating enzyme. Surprisingly, five ta-siRNA-generating transcripts were identified as targets of miR173 or miR390. Rather than functioning as negative regulators, miR173- and miR390-guided cleavage was shown to set the 21-nucleotide phase for ta-siRNA precursor processing. These data support a model in which miRNA-guided formation of a 5′ or 3′ terminus within pre-ta-siRNA transcripts, followed by RDR6-dependent formation of dsRNA and Dicer-like processing, yields phased ta-siRNAs that negatively regulate other genes.

[21] SMOOT M E, ONO K, RUSCHEINSKI J, WANG P L, IDEKER T . Cytoscape 2.8: new features for data integration and network visualization
Bioinformatics, 2011,27(3):431-432.
DOI:10.1093/bioinformatics/btq675URLPMID:21149340 [本文引用: 1]
ABSTRACTSummary: Cytoscape is a popular bioinformatics package for biological network visualization and data integration. Version 2.8 introduces two powerful new features--Custom Node Graphics and Attribute Equations--which can be used jointly to greatly enhance Cytoscape's data integration and visualization capabilities. Custom Node Graphics allow an image to be projected onto a node, including images generated dynamically or at remote locations. Attribute Equations provide Cytoscape with spreadsheet-like functionality in which the value of an attribute is computed dynamically as a function of other attributes and network properties.Availability and implementation: Cytoscape is a desktop Java application released under the Library Gnu Public License (LGPL). Binary install bundles and source code for Cytoscape 2.8 are available for download from http://cytoscape.org.Contact: msmoot@ucsd.edu

[22] CHEN C, RIDZON D A, BROOMER A J, ZHOU Z, LEE D H, NGUYEN J T, BARBISIN M, XU N L, MAHUVAKAR V R, ANDERSEN M R, LAO K Q, LIVAK K J, GUEGLER K J . Real-time quantification of microRNAs by stem-loop RT-PCR
Nucleic Acids Research, 2005,33(20):e179.
DOI:10.1093/nar/gni178URLPMID:16314309 [本文引用: 1]
A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.

[23] LUCAS K J, ZHAO B, LIU S, RAIKHEL A S . Regulation of physiological processes by microRNAs in insects
Current Opinion in Insect Science, 2015,11:1-7.
DOI:10.1016/j.cois.2015.06.004URLPMID:26251827 [本文引用: 1]
MicroRNAs (miRNAs) are small non-coding RNAs that function in gene regulatory processes in plants and animals by targeting sites within messenger RNA. In insects, miRNAs have been shown to regulate a variety of physiological processes throughout insect development, including molting, metamorphosis, oogenesis, embryogenesis, behavior and host athogen interactions. The roles of miRNAs in the model organism, Drosophila melanogaster, have been studied extensively due to the conserved nature of miRNA function among highly divergent species. However, seeking to understand miRNA function in non-drosophilid insect species has become a growing trend in insect science. Here, we highlight the recent discoveries regarding miRNA function in insect physiology and development.

[24] ZHANG Y, ZHOU X, GE X, LI M, JIA S, YANG X, KAN Y, MIAO X, ZHAO G, LI F, HUANG Y . Insect-specific microRNA involved in the development of the silkworm Bombyx mori.
PLoS ONE, 2009,4(3):e4677.
DOI:10.1371/journal.pone.0004677URLPMID:2650705 [本文引用: 1]
MicroRNAs (miRNAs) are endogenous non-coding genes that participate in post-transcription regulation by either degrading mRNA or blocking its translation. It is considered to be very important in regulating insect development and metamorphosis. We conducted a large-scale screening for miRNA genes in the silkwormBombyx moriusing sequence-by-synthesis (SBS) deep sequencing of mixed RNAs from egg, larval, pupal, and adult stages. Of 2,227,930 SBS tags, 1,144,485 ranged from 17 to 25 nt, corresponding to 256,604 unique tags. Among these non-redundant tags, 95,184 were matched to the silkworm genome. We identified 3,750 miRNA candidate genes using a computational pipeline combining RNAfold and TripletSVM algorithms. We confirmed 354 miRNA genes using miRNA microarrays and then performed expression profile analysis on these miRNAs for all developmental stages. While 106 miRNAs were expressed in all stages, 248 miRNAs were egg- and pupa-specific, suggesting that insect miRNAs play a significant role in embryogenesis and metamorphosis. We selected eight miRNAs for quantitative RT-PCR analysis; six of these were consistent with our microarray results. In addition, we searched for orthologous miRNA genes in mammals, a nematode, and other insects and found that most silkworm miRNAs are conserved in insects, whereas only a small number of silkworm miRNAs has orthologs in mammals and the nematode. These results suggest that there are many miRNAs unique to insects.

[25] LIU S, GAO S, ZHANG D, YIN J, XIANG Z, XIA Q . MicroRNAs show diverse and dynamic expression patterns in multiple tissues of Bombyx mori.
BMC Genomics, 2010,11:85.
DOI:10.1186/1471-2164-11-85URLPMID:2835664 [本文引用: 2]
pAbstract/p pBackground/p pMicroRNAs (miRNAs) repress target genes at the post-transcriptional level, and function in the development and cell-lineage pathways of host species. Tissue-specific expression of miRNAs is highly relevant to their physiological roles in the corresponding tissues. However, to date, few miRNAs have been spatially identified in the silkworm./p pResults/p pWe establish for the first time the spatial expression patterns of nearly 100 miRNAs in multiple normal tissues (organs) of itBombyx mori /itfemales and males using microarray and Northern-blotting analyses. In all, only 10 miRNAs were universally distributed (including bmo-let-7 and bmo-bantam), while the majority were expressed exclusively or preferentially in specific tissue types (e.g., bmo-miR-275 and bmo-miR-1). Additionally, we examined the developmental patterns of miRNA expression during metamorphosis of the body wall, silk glands, midgut and fat body. In total, 63 miRNAs displayed significant alterations in abundance in at least 1 tissue during the developmental transition from larvae to pupae (e.g., bmo-miR-263b and bmo-miR-124). Expression patterns of five miRNAs were significantly increased during metamorphosis in all four tissues (e.g., bmo-miR-275 and bmo-miR-305), and two miRNA pairs, bmo-miR-10b-3p/5p and bmo-miR-281-3p/5p, showed coordinate expression./p pConclusions/p pIn this study, we conducted preliminary spatial measurements of several miRNAs in the silkworm. Periods of rapid morphological change were associated with alterations in miRNA expression patterns in the body wall, silk glands, midgut and fat body during metamorphosis. Accordingly, we propose that corresponding ubiquitous or tissue-specific expression of miRNAs supports their critical roles in tissue specification. These results should facilitate future functional analyses./p

[26] LIANG P, FENG B, ZHOU X G, GAO X W . Identification and developmental profiling of microRNAs in diamondback moth,Plutella xylostella(L.).
PLoS ONE, 2013,8(11):e78787.
DOI:10.1371/journal.pone.0078787URLPMID:3827265 [本文引用: 1]
MicroRNAs (miRNAs) are a group of small RNAs involved in various biological processes through negative regulation of mRNAs at the post-transcriptional level. Although miRNA profiles have been documented in over two dozen insect species, few are agricultural pests. In this study, both conserved and novel miRNAs in the diamondback moth,Plutella xylostellaL., a devastating insect pest of cruciferous crops worldwide, were documented. High-throughput sequencing of a small RNA library constructed from a mixed life stages ofP. xylostella,including eggs, 1st to 4th (last) instar larvae, pupae and adults, identified 384 miRNAs, of which 174 wereP. xylostellaspecific. In addition, temporal expressions of 234 miRNAs at various developmental stages were investigated using a customized microarray analysis. Among the 91 differentially expressed miRNAs, qRT-PCR analysis was used to validate highly expressed miRNAs at each stage. The combined results not only systematically document miRNA profiles in an agriculturally important insect pest, but also provide molecular targets for future functional analysis and, ultimately, genetic-based pest control practice.

[27] 陈璇, 俞晓敏, 郑火青, 蔡亦梅, 胡福良 . 西方蜜蜂( Apis mellifera L.) sRNA的富集与文库检测
中国农业科学, 2009,42(8):2943-2948.
Magsci [本文引用: 1]
<P><FONT face=Verdana>【目的】提取及扩增蜜蜂（Apis mellifera L）sRNA,并构建文库检测富集结果是否满足高通量测序研究要求。【方法】取蜜蜂3个级型不同发育阶段个体作为材料,分别提取总RNA后混合,从中分离出15～40nt的sRNA,反转成cDNA后构建文库,进行蓝白斑筛选。挑选288个单克隆进行测序,对测序结果进行分析。【结果】有效序列为214条,插入的cDNA片段大小范围为15～39 bp。其中,sme-miR-71c miRNA 65条,ncRNA（包括tm-RNA、intron_ghI、5.8s rRNA）5条,tRNA 28条,siRNA及其他sRNA 33条, CDS 1条,未知序列82条。【结论】本实验采用的方法能有效富集蜜蜂sRNA,能够满足高通量测序从中识别出蜜蜂miRNA的研究。<BR></FONT></P>
CHEN X, YU X M, ZHENG H Q, CAI Y M, HU F L . Separation and enrichment of sRNAs from honeybee (Apis mellifera L.) and its quality detection by library construction.
Scientia Agricultura Sinica, 2009,42(8):2943-2948. (in Chinese)
Magsci [本文引用: 1]
<P><FONT face=Verdana>【目的】提取及扩增蜜蜂（Apis mellifera L）sRNA,并构建文库检测富集结果是否满足高通量测序研究要求。【方法】取蜜蜂3个级型不同发育阶段个体作为材料,分别提取总RNA后混合,从中分离出15～40nt的sRNA,反转成cDNA后构建文库,进行蓝白斑筛选。挑选288个单克隆进行测序,对测序结果进行分析。【结果】有效序列为214条,插入的cDNA片段大小范围为15～39 bp。其中,sme-miR-71c miRNA 65条,ncRNA（包括tm-RNA、intron_ghI、5.8s rRNA）5条,tRNA 28条,siRNA及其他sRNA 33条, CDS 1条,未知序列82条。【结论】本实验采用的方法能有效富集蜜蜂sRNA,能够满足高通量测序从中识别出蜜蜂miRNA的研究。<BR></FONT></P>

[28] 陈璇 . 蜜蜂( Apis mellifera) microRNA的全基因组挖掘及在雌性蜜蜂级型分化关键时期转录组水平调控作用
[D]. 杭州: 浙江大学, 2012.
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CHEN X . Genome-wide identification of microRNAs and their regulation of transcriptome on female caste determination of honey bee (Apis mellifera)
[D]. Hangzhou: Zhejiang University, 2012. ( in Chinese)
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[29] 汝玉涛, 王勇, 周敬林, 王德意, 马月月, 姜义仁, 高清, 秦利 . 蜕皮激素受体和超气门蛋白基因在柞蚕发育过程及激素诱导后的表达模式
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[D]. 重庆: 西南大学, 2009.
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SHU X . Cloning and expression analysis of genes encoding ecdysis triggering hormone and its receptor in the silkworm, Bombyx mori
[D]. Chongqing: Southwest University, 2009. ( in Chinese)
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[31] 周艳河 . 白纹伊蚊中肠特异性高表达miRNA-miR-281对登革病毒复制的调节作用
[D]. 广州: 南方医科大学, 2014.
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ZHOU Y H . Dengue virus replication is regulated by miR-281: an abundant midgut-specific miRNA of vector mosquito Aedes albopictus
[D]. Guangzhou: Southern Medical University, 2014. ( in Chinese)
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[32] 熊慧萍 . 位于可变内含子区的果蝇microRNA-281-1/2基因转录和启动子分析
[D]. 南京: 南京农业大学, 2008.
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XIONG H P . Transcription and promoter analysis of Drosophila intronic microRNA-281-1/2 located in alternative spliced region
[D]. Nanjing: Nanjing Agricultural University, 2008. (in Chinese)
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Abstract The Drosophila Bithorax Complex encodes three well-characterized homeodomain proteins that direct segment identity, as well as several noncoding RNAs of unknown function. Here, we analyze the iab-4 locus, which produces the microRNAs iab-4-5p and iab-4-3p. iab-4 is analogous to miR-196 in vertebrate Hox clusters. Previous studies demonstrate that miR-196 interacts with the Hoxb8 3' untranslated region. Evidence is presented that miR-iab-4-5p directly inhibits Ubx activity in vivo. Ectopic expression of mir-iab-4-5p attenuates endogenous Ubx protein accumulation and induces a classical homeotic mutant phenotype: the transformation of halteres into wings. These findings provide the first evidence for a noncoding homeotic gene and raise the possibility that other such genes occur within the Bithorax complex. We also discuss the regulation of mir-iab-4 expression during development.

[34] TAMáSI V, MONOSTORY K, PROUGH R A, FALUS A . Role of xenobiotic metabolism in cancer: involvement of transcriptional and miRNA regulation of P450s
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DOI:10.1007/s00018-010-0600-7URLPMID:21184128 [本文引用: 1]
Abstract Cytochrome P450 enzymes (P450s) are important targets in cancer, due to their role in xenobiotic metabolism. Since P450s are the "bridges" between the environment and our body, their function can be linked in many ways to carcinogenesis: they activate dietary and environmental components to ultimate carcinogens (i), the cancer tissue maintains its drug resistance with altered expression of P450s (ii), P450s metabolize (sometimes activate) drugs used for cancer treatment (iii) and they are potential targets for anticancer therapy (iiii). These highly polymorphic enzymes are regulated at multiple molecular levels. Regulation is as important as genetic difference in the existing individual variability in P450 activity. In this review, examples of the transcriptional (DNA methylation, histone modification, modulation by xenosensors) and post-transcriptional (miRNA) regulation will be presented and thereby introduce potential molecular targets at which the metabolism of anticancer drugs, the elimination of cancerogenes or the progress of carcinogenesis could be affected.

[35] 黄献彬 . 小菜蛾解毒代谢相关miRNA鉴定及表达分析
[D]. 福州: 福建农林大学, 2016.
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HUANG X B . Identification and analysis of miRNAs involved in detoxification in the diamondback moth, Plutella xylostella
[D]. Fuzhou: Fujian Agriculture and Forestry University, 2016. ( in Chinese)
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