Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (1): 201-212.doi: 10.3864/j.issn.0578-1752.2020.01.019

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

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

Yu DU,DingDing ZHOU,JieQi WAN,JiaXuan LU,XiaoXue FAN,YuanChan FAN,Heng CHEN,CuiLing XIONG,YanZhen ZHENG,ZhongMin FU,GuoJun XU,DaFu CHEN,Rui GUO()   

  1. College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002
  • Received:2019-07-09 Accepted:2019-09-02 Online:2020-01-01 Published:2020-01-19
  • Contact: Rui GUO E-mail:ruiguo@fafu.edu.cn

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Abstract:

【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, P13K-Akt, Wnt, cAMP, FoxO, Hippo, mTOR, 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, PI3K-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 DEG was enriched in several signaling pathways. Moreover, regulation network analysis showed that 54 up-regulated genes and 44 down-regulated genes were targets of ame-miR-6001-3p, respectively; these up-regulated genes were enriched in 43 pathways including inositol phosphate metabolism, Hippo signaling pathway, glutathione metabolism and insulin signaling pathway, while these down-regulated genes were enriched in 20 pathways including Hippo signaling pathway, metabolic pathways, glutathione metabolism and arachidonic acid metabolism. Moreover, RT-qPCR result showed that the variation trend of six randomly selected DEGs were consistent with that in sequencing data, confirming the reliability of DEGs. Finally, ame-miR-6001-3p was definitely expressed and significantly down-regulated in Am10.【Conclusion】In this work, the expression pattern of DEGs and the regulation network between DEGs and ame-miR-6001-3p as well as the potential role of DEGs during the developmental process of A. m. ligustica worker’s midgut were deeply analyzed. The results revealed that the DEGs may participate in various signaling pathways including TGF-beta, Wnt, Hippo, Notch, PI3K-Akt, mTOR, AMPK, NF-κB signaling pathways, thus affecting the growth and development as well as immune defense of the midgut; DEGs were likely to regulate several signaling pathways such as insulin signaling pathway during the midgut development via formation regulation networks with significantly down-regulated ame-miR-6001-3p.

Key words: Apis mellifera ligustica, midgut, development, differentially expressed gene (DEG), competitive endogenous RNA, regulation network

Table 1

Primers used in this study"

引物名称Primer name 引物序列Primer sequence
XM_016912284.1-F AGCGATTGAAGATGTGCC
XM_016912284.1-R ACGACAGGTGAAACCGAA
TCONS_00023605-F GCAGTGACCTTGGCGTATT
TCONS_00023605-R CATTCTGACCGTTGATTTGC
XM_016912389.1-F TAAGGTGCTGCTACCACTGC
XM_016912389.1-R GCTCTGTTAGGGTCGCATCT
TCONS_00001615-F GTGGACGACACACGATTCT
TCONS_00001615-R CCAATAACAGCGACACACG
XM_001123053.4-F TTGATGCTGTGAAGGCTG
XM_001123053.4-R AGGCGAACTGTAAACGGTT
XM_016915022.1-F ATCAACAACACCCACACCT
XM_016915022.1-R AGGAACGGATTCGCTGTA
Actin-F CACTCCTGCTATGTATGTCGC
Actin-R GGCAAAGCGTATCCTTCA
ame-miR-6001-3p-loop CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTAGTGGTA
ame-miR-6001-3p-F ACACTCCAGCTGGGTTCTCTTTGGTTGT
ame-miR-6001-3p-R CTCAACTGGTGTCGTGGAGTCG
U6-F GTTAGGCTTTGACGATTTCG
U6-R GGCATTTCTCCACCAGGTA

Fig. 1

Volcano plot of DEGs during the developmental process of A. m. ligustica worker’s midgut"

Fig. 2

GO classification of DEGs during the developmental process of A. m. ligustica worker’s midgut"

Fig. 3

Expression clustering of DEGs enriched in AMPK, PI3K-Akt, Hippo signaling pathway and endocytosis"

Fig. 4

Relationship networks between 13 signaling pathways and related DEGs in A. m. ligustica worker’s midgut"

Fig. 5

Regulation networks between DEGs and ame-miR-6001-3p in A. m. ligustica worker’s midgut"

Fig. 6

Validation of DEGs and ame-miR-6001-3p"

[1] COON K L, VOGEL K J, BROWN M R, STRAND M R . Mosquitoes rely on their gut microbiota for development. Molecular Ecology, 2014,23(11):2727-2739.
[2] CHOUAIA B, ROSSI P, MONTAGNA M, RICCI I, CROTTI E, DAMIANI C, EPIS S, FAYE I, SAGNON N, ALMA A, FAVIA G, DAFFONCHIO D, BANDI C . Molecular evidence for multiple infections as revealed by typing of Asaia bacterial symbionts of four mosquito species. Applied and Environmental Microbiology, 2010,76(22):7444-7450.
[3] STORELLI G, DEFAYE A, ERKOSAR B, HOLS P, ROYET J, LEULIER F . Lactobacillus plantarum promotes Drosophila systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing. Cell Metabolism, 2011,14(3):403-414.
[4] ZHENG H, POWELL J E, STEELE M I, DIETRICH C, MORAN N A . Honeybee gut microbiota promotes host weight gain via bacterial metabolism and hormonal signaling. Proceedings of the National Academy of Sciences of the United States of America, 2017,114(18):4775-4780.
[5] 冯倩倩 . 意大利蜜蜂春繁阶段代用花粉中VA、VE适宜水平的研究[D]. 泰安: 山东农业大学, 2012.
FENG Q Q . Optimal level of VA and VE in pollen substitutes of Apis mellifera ligustica during the period of spring multiplication[D]. Taian: Shandong Agricultural University, 2012. (in Chinese)
[6] 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, YIN W X, ZHENG Y Z, FU Z M, CHEN D F . Trend analysis for differentially expressed genes in the 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)
[7] 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. 中国农业科学, 2018,51(21):4197-4209.
GUO R, DU Y, XIONG C L, ZHENG Y Z, FU Z M, XU G J, WANG H P, CHEN H Z, GENG S H, ZHOU D D, SHI C Y, ZHAO H X, CHEN D F . Differentially expressed microRNA and their regulation networks during the developmental process of Apis mellifera ligustica larval gut. Scientia Agricultura Sinica, 2018,51(21):4197-4209. (in Chinese)
[8] MELLO T R P, ALEIXO A C, PINHEIRO D G, NUNES F M F, BITONDI M M G, HARTFELDER K, BARCHUK A R, SIMÕES Z L P . Developmental regulation of ecdysone receptor (EcR) and EcR-controlled gene expression during pharate-adult development of honeybees ( Apis mellifera). Frontiers in Genetics, 2014,5:445.
[9] COLLINS D H, MOHORIANU I, BECKERS M, MOULTON V, DALMAY T, BOURKE A F . MicroRNAs associated with caste determination and differentiation in a primitively eusocial insect. Scientific Reports, 2017,7:45674.
[10] 郭睿, 耿四海, 熊翠玲, 郑燕珍, 付中民, 王海朋, 杜宇, 童新宇, 赵红霞, 陈大福 . 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. 中国农业科学, 2018,51(18):3600-3613.
GUO R, GENG S H, XIONG C L, ZHENG Y Z, FU Z M, WANG H P, DU Y, TONG X Y, ZHAO H X, CHEN D F . Differential expression analysis of long non-coding RNAs during the developmental process of Apis mellifera ligustica worker’s midgut. Scientia Agricultura Sinica, 2018,51(18):3600-3613. (in Chinese)
[11] 郭睿, 陈华枝, 熊翠玲, 郑燕珍, 付中民, 徐国钧, 杜宇, 王海朋, 耿四海, 周丁丁, 刘思亚, 陈大福 . 意大利蜜蜂工蜂中肠发育过程中的差异表达环状RNA及其调控网络分析. 中国农业科学, 2018,51(23):4575-4590.
GUO R, CHEN H Z, XIONG C L, ZHENG Y Z, FU Z M, XU G J, DU Y, WANG H P, GENG S H, ZHOU D D, LIU S Y, CHEN D F . Analysis of differentially expressed circular RNAs and their regulation networks during the developmental process of Apis mellifera ligustica worker’s midgut. Scientia Agricultura Sinica, 2018,51(23):4575-4590. (in Chinese)
[12] HUANG Q, CHEN Y P, WANG R W, CHENG S, EVANS J D . Host-parasite interactions and purifying selection in a microsporidian parasite of honey bees. PLoS ONE, 2016,11(2):e0147549.
[13] HUANG W F, SOLTER L F . Comparative development and tissue tropism of Nosema apis and Nosema ceranae. Journal of Invertebrate Pathology, 2013,113(1):35-41.
[14] SMOOT M E, ONO K, RUSCHEINSKI J, WANG P L, IDEKER T . Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics (Oxford, England), 2011,27(3):431-432.
[15] 郭睿, 杜宇, 童新宇, 熊翠玲, 郑燕珍, 徐国钧, 王海朋, 耿四海, 周丁丁, 郭意龙, 吴素珍, 陈大福 . 意大利蜜蜂幼虫肠道在球囊菌侵染前期的差异表达microRNA及其调控网络. 中国农业科学, 2019,52(1):166-180.
GUO R, DU Y, TONG X Y, XIONG C L, ZHENG Y Z, XU G J, WANG H P, GENG S H, ZHOU D D, GUO Y L, WU S Z, CHEN D F . Differentially expressed microRNAs and their regulation networks in Apis mellifera ligustica larval gut during the early stage of Ascosphaera apis infection. Scientia Agricultura Sinica, 2019,52(1):166-180. (in Chinese)
[16] MARTINSON V G, MOY J, MORAN N A . Establishment of characteristic gut bacteria during development of the honeybee worker. Applied and Environmental Microbiology, 2012,78(8):2830-2840.
[17] KWONG W K, MANCENIDO A L, MORAN N A . Immune system stimulation by the native gut microbiota of honey bees. Royal Society Open Science, 2017,4(2):170003.
[18] CHEN D, GUO R, XU X, XIONG C, LIANG Q, ZHENG Y, LUO Q, ZHANG Z, HUANG Z, KUMAR D, XI W, 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.
[19] 郭睿, 杜宇, 周倪红, 刘思亚, 熊翠玲, 郑燕珍, 付中民, 徐国钧, 王海朋, 耿四海, 周丁丁, 陈大福 . 意大利蜜蜂幼虫肠道在球囊菌胁迫后期的差异表达微小RNA及其靶基因分析. 昆虫学报, 2019,62(1):49-60.
GUO R, DU Y, ZHOU N H, LIU S Y, XIONG C L, ZHENG Y Z, FU Z M, XU G J, WANG H P, GENG S H, ZHOU D D, CHEN D F . Comprehensive analysis of differentially expressed microRNAs and their target genes in the larval gut of Apis mellifera ligustica during the late stage of Ascosphaera apis stress. Acta Entomologica Sinica, 2019,62(1):49-60. (in Chinese)
[20] BARRY E R, CAMARGO F D . The Hippo superhighway: signaling crossroads converging on the Hippo/Yap pathway in stem cells and development. Current Opinion in Cell Biology, 2013,25(2):247-253.
[21] 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)
[22] 宁越, 米雪, 陈星伊, 邵建航, 昝林森 . SMAD1基因的沉默和过表达及对秦川牛原代成肌细胞生肌的影响. 中国农业科学, 2019,52(10):1818-1829.
NING Y, MI X, CHEN X Y, SHAO J H, ZAN L S . Silencing and overexpressing SMAD family member 1 (SMAD1) gene and its effect on myogenesis in primary myoblast of Qinchuan cattle(Bos taurus). Scientia Agricultura Sinica, 2019,52(10):1818-1829. (in Chinese)
[23] BRUMMEL T, ABDOLLAH S, HAERRY T E, SHIMELL M J, MERRIAM J, RAFTERY L, WRANA J L, O’CONNOR M B . The Drosophila activin receptor baboon signals through dSmad2 and controls cell proliferation but not patterning during larval development. Genes and Development, 1999,13(1):98-111.
[24] 张振, 卢忠燕 . 家蚕类胰岛素信号通路及其功能的研究进展. 蚕学通讯, 2014,34(2):22-26.
ZHANG Z, LU Z Y . Research progress on insulin signaling pathway and its function in silkworm. Newsletter of Sericultural Science, 2014,34(2):22-26. (in Chinese)
[25] GAUTAM S, ISHRAT N, YADAV P, SINGH R, NARENDER T, SRIVASTAVA A K . 4-Hydroxyisoleucine attenuates the inflammation- mediated insulin resistance by the activation of AMPK and suppression of SOCS-3 coimmunoprecipitation with both the IR-β subunit as well as IRS-1. Molecular and Cellular Biochemistry, 2016,414(1/2):95-104.
[26] NEUFELD T P . Body building: regulation of shape and size by PI3K/TOR signaling during development. Mechanisms of Development, 2003,120(11):1283-1296.
[27] KAUR S, SASSANO A, DOLNIAK B, JOSHI S, MAJCHRZAK- KITA B, BAKER D P, HAY N, FISH E N, PLATANIAS L C . Role of the Akt pathway in mRNA translation of interferon-stimulated genes. Proceedings of the National Academy of Sciences of the United States of America, 2008,105(12):4808-4813.
[28] LIU S, LUCAS K J, ROY S, HA J, RAIKHEL A S . Mosquito-specific microRNA-1174 targets serine hydroxymethyltransferase to control key functions in the gut. Proceedings of the National Academy of Sciences of the United States of America, 2014,111(40):14460-14465.
[29] 熊翠玲, 杜宇, 陈大福, 郑燕珍, 付中民, 王海朋, 耿四海, 陈华枝, 周丁丁, 吴素珍, 石彩云, 郭睿 . 意大利蜜蜂幼虫肠道的miRNAs的生物信息学预测及分析. 应用昆虫学报, 2018,55(6):1023-1033.
XIONG C L, DU Y, CHEN D F, ZHENG Y Z, FU Z M, WANG H P, GENG S H, CHEN H Z, ZHOU D D, WU S Z, SHI C Y, GUO R . Bioinformatic prediction and analysis of miRNAs in the Apis mellifera ligustica larval gut. Chinese Journal of Applied Entomology, 2018,55(6):1023-1033. (in Chinese)
[30] KARRENTH F A, TAY Y, PERNA D, ALA U, TAN S M, RUST A G, DENICOLA G, WEBSTER K A, WEISS D PEREZ-MANCERA P A, KRAUTHAMMER M, HALABAN R, PROVERO P, ADAMS D J, TUVESON D A, PANDOLFI P P . In vivo identification of tumor-suppressive PTEN ceRNAs in an oncogenic BRAF-induced mouse model of melanoma. . Cell, 2011,147(2):382-395.
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