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

doi:10.3864/j.issn.0578-1752.2018.21.018

摘要/Abstract

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

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.

Rui GUO,Yu DU,CuiLing XIONG,YanZhen ZHENG,ZhongMin FU,GuoJun XU,HaiPeng WANG,HuaZhi CHEN,SiHai GENG,DingDing ZHOU,CaiYun SHI,HongXia ZHAO,DaFu CHEN. Differentially Expressed MicroRNA and Their Regulation Networks During the Developmental Process of Apis mellifera ligustica Larval Gut[J]. Scientia Agricultura Sinica, 2018, 51(21): 4197-4209.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2018.21.018

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参考文献 35

[1]	AZZAM G, SMIBERT P, LAI E C, LIU J L . Drosophila Argonaute 1 and its miRNA biogenesis partners are required for oocyte formation and germline cell division. Developmental Biology, 2012,365(2):384-394. doi: 10.1016/j.ydbio.2012.03.005 pmid: 3763516
[2]	ASGARI S . MicroRNA functions in insects. Insect Biochemistry and Molecular Biology, 2013,43(4):388-397. doi: 10.1016/j.ibmb.2012.10.005
[3]	XU L, HU Y G, CAO Y, LI J R, MA L G, LI Y, QI Y J . An expression atlas of miRNAs in Arabidopsis thaliana. Science China Life Sciences, 2018,61(2):178-189.
[4]	LIU M, YU H, ZHAO G, HUANG Q, LU Y, OUYANG B . Profiling of drought-responsive microRNA and mRNA in tomato using high-throughput sequencing. BMC Genomics, 2017,18(1):481. doi: 10.1186/s12864-017-3869-1 pmid: 28651543
[5]	YU Z Q, GAO X L, LIU C N, LV X P, ZHENG S M . Analysis of microRNA expression profile in specific pathogen-free chickens in response to reticuloendotheliosis virus infection. Applied Microbiology and Biotechnology, 2017,101(7):2767-2777. doi: 10.1007/s00253-016-8060-0 pmid: 28032193
[6]	OJHA C R, RODRIGUEZ M, DEVER S M, MUKHOPADHYAY R, EL-HAGE N . Mammalian microRNA: an important modulator of host-pathogen interactions in human viral infections. Journal of Biomedical Science, 2016,23(1):74. doi: 10.1186/s12929-016-0292-x pmid: 27784307
[7]	The Honeybee Genome Sequencing Consortium. Insights into social insects from the genome of the honeybee Apis mellifera. Nature, 2006,443(7114):931-949.
[8]	刘芳 . 意蜂哺育蜂与采集蜂头部mRNAs与miRNAs表达谱Solexa测序比较分析及其调控网络研究[D]. 杭州: 浙江大学, 2012.
	LIU F . Integrating of Solexa high-abundance mRNAs and miRNAs in Apis mellifera: comparison between nurses and foragers to identify regulatory network[D]. Hangzhou: Zhejiang University, 2012. (in Chinese)
[9]	郭昱, 苏松坤, 陈盛禄, 张少吾, 陈润生 . LncRNA在蜜蜂级型分化中的功能研究. 生物化学与生物物理进展, 2015,42(8):750-757.
	GUO Y, SU S K, CHEN S L, ZHANG S W, CHEN R S . The function of lncRNAs in the caste determination of the honeybee. Progress in Biochemistry and Biophysics, 2015,42(8):750-757. (in Chinese)
[10]	ASHBY R, FORÊT S, SEARLE I, MALESZKA R . MicroRNAs in honey bee caste determination. Scientific Reports, 2016,6:18794. doi: 10.1038/srep18794 pmid: 4704047
[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.
	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)
[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.
	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)
[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.
	CHANG W . Diversity of symbiotic bacteria in honeybee alimentary tract[D]. Fuzhou: Fujian Agriculture and Forestry University, 2010. ( in Chinese)
[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.
	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)
[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.
	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)
[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.
[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.2017.04.005
	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.2017.04.005
[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.1001-4942.2009.11.037
	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.1001-4942.2009.11.037
[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/gkr688 pmid: 21911355
[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.2005.04.004 pmid: 15851028
[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/btq675 pmid: 21149340
[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/gni178 pmid: 16314309
[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.2015.06.004 pmid: 26251827
[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.0004677 pmid: 2650705
[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: 10.1186/1471-2164-11-85 pmid: 2835664
[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.0078787 pmid: 3827265
[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.
	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)
[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.
	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)
[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. 蚕业科学, 2017(4):594-602.
	RU Y T, WANG Y, ZHOU J L, WANG D Y, MA Y Y, JIANG Y R, GAO Q, QIN L . The expression patterns of ecdysone receptor and ultraspiracle genes inAntheraea pernyi during development and hormone-induced process. Science of Sericulture, 2017(4):594-602. (in Chinese)
[30]	舒旭 . 家蚕蜕皮触发激素及其受体基因的克隆和表达分析[D]. 重庆: 西南大学, 2009.
	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)
[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.
	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)
[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.
	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)
[33]	RONSHAUGEN M, BIEMAR F, PIEL J, LEVINE M, LAI E C . The Drosophila microRNA iab-4 causes a dominant homeotic transformation of halteres to wings. Genes and Development, 2005,19(24):2947-2952. doi: 10.1101/gad.1372505 pmid: 16357215
[34]	TAMáSI V, MONOSTORY K, PROUGH R A, FALUS A . Role of xenobiotic metabolism in cancer: involvement of transcriptional and miRNA regulation of P450s. Cellular and Molecular Life Sciences, 2011,68(7):1131-1146. doi: 10.1007/s00018-010-0600-7 pmid: 21184128
[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.
	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)

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%)