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

doi:10.3864/j.issn.0578-1752.2020.01.019

Abstract

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

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. Profiling and Regulation Network of Differentially Expressed Genes During the Development Process of Apis mellifera ligustica Worker’s Midgut[J].Scientia Agricultura Sinica, 2020, 53(1): 201-212.

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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

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

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