意大利蜜蜂响应东方蜜蜂微孢子虫胁迫的免疫应答

doi:10.3864/j.issn.0578-1752.2019.17.014

摘要/Abstract

摘要：

【目的】通过对意大利蜜蜂（Apis mellifera ligustica,简称意蜂）工蜂中肠响应东方蜜蜂微孢子虫（Nosema ceranae）胁迫的差异表达基因（differentially expressed gene,DEG）及免疫通路进行深入细致的分析,揭示宿主的细胞和体液免疫应答,为关键免疫应答基因的筛选及功能研究打下基础。【方法】基于前期获得的正常及东方蜜蜂微孢子虫胁迫的意蜂7日龄和10日龄工蜂中肠（Am7CK、Am7T、Am10CK、Am10T）转录组数据,按照FDR≤1,P≤0.05和|log₂fold change|≥1的标准筛选出各组的DEG,利用相关生物信息学软件对DEG进行Pearson相关性分析、Venn分析、GO分类和KEGG代谢通路富集分析,进而对免疫通路富集基因进行统计和分析,利用实时荧光定量PCR（real-time quantitative PCR,RT-qPCR）验证转录组数据的可靠性。【结果】 差异分析结果显示,Am7CK vs Am7T比较组包含472个上调基因和385个下调基因;Am10CK vs Am10T比较组包含611个上调基因和360个下调基因。Venn分析结果显示,两个比较组特有的DEG分别为739和853个,共有的DEG为118个。GO分类结果显示,Am7CK vs Am7T比较组中上调和下调基因分别涉及23和29个功能条目,其中富集上调基因数最多的前5位分别为结合、催化活性、代谢进程、细胞进程和单组织进程;富集下调基因数最多的前5位分别为代谢进程、单组织进程、催化活性、细胞进程和结合。Am10CK vs Am10T比较组中上调和下调基因分别涉及36和26个功能条目,其中富集上调基因数最多的前5位分别为单组织进程、结合、细胞进程、催化活性和代谢活性;富集下调基因数最多的前5位分别为结合、细胞进程、催化活性、代谢进程和单组织进程。KEGG代谢通路富集分析结果显示,Am7CK vs Am7T比较组中上调和下调基因分别富集在38和33条代谢通路,富集上调基因数最多的前5条分别为胆汁分泌、内质网蛋白加工、泛素介导的蛋白水解、PI3K-Akt信号通路和神经营养因子信号通路;富集下调基因数最多的前5条分别为胞质DNA传感通路、嘌呤代谢、嘧啶代谢、RNA聚合酶和核糖体;涉及泛素介导的蛋白水解等3条细胞免疫通路,以及PI3K-Akt信号通路等7条体液免疫通路。Am10CK vs Am10T比较组中上调和下调基因分别富集在54和43条代谢通路,富集上调基因数最多的前5条分别为Hippo信号通路、药物代谢-细胞色素P450、P450对外源物质的代谢、泛素介导的蛋白水解和鞘脂类代谢;富集下调基因数最多的前5条分别为mRNA监测、鞘脂类信号通路、果糖和甘露糖代谢、半乳糖代谢和鞘脂类代谢;涉及泛素介导的蛋白水解等7条细胞免疫通路,以及NF-κB信号通路等2条体液免疫通路。RT-qPCR验证结果显示6个随机挑选的DEG的表达量变化趋势与测序结果一致,证实了本研究中测序数据的可靠性。进一步分析发现意蜂7日龄和10日龄工蜂中肠的NF-κB信号通路均被东方蜜蜂微孢子虫激活,随即启动了3种抗菌肽apidaecin、defensin-1和hymenoptaecin的合成,体现了它们在宿主抵御东方蜜蜂微孢子虫入侵中的重要性。【结论】在转录组水平解析了意蜂工蜂中肠对东方蜜蜂微孢子虫的免疫应答,揭示宿主在胁迫前期同时作出细胞和体液免疫应答,前者可能在抵御病原入侵方面发挥主要作用;宿主的细胞免疫在胁迫后期持续增强,但体液免疫较大程度地减弱;泛素介导的蛋白水解通路及富集DEG,NF-κB信号通路及富集DEG,以及抗菌肽编码基因apidaecin、defensin-1和hymenoptaecin可能在意蜂工蜂对东方蜜蜂微孢子虫的免疫应答中起到关键作用。

关键词: 意大利蜜蜂, 东方蜜蜂微孢子虫, 中肠, 免疫应答, 细胞免疫, 体液免疫

Abstract:

【Objective】 The objective of this study is to reveal the cellular and humoral immune responses of Apis mellifera ligusitca worker’s midgut to Nosema ceranae stress, and to provide a basis for screening and functional study of key immune response genes by deep investigation of host differentially expressed genes (DEGs) and immune pathways.【Method】 Base on the previously obtained transcriptome datasets of normal and N. ceranae-stressed midguts of A. m. ligustica 7- and 10-day-old workers’ midgut samples (Am7CK, Am7T, Am10CK and Am10T), DEGs in each comparison group were filtered out following the standard FDR≤1, P≤0.05 and |log₂fold change|≥1. Additionally, Pearson correlations, Venn analysis, GO classification and KEGG pathway enrichment analysis were conducted by using related bioinformatic softwares. Further, DEGs enriched in immune pathways were summarized and analyzed. Finally, the reliability of transcriptome data was verified via real-time quantitative PCR (RT-qPCR).【Result】 Differential expression analysis showed that there were 472 up-regulated and 385 down-regulated genes in Am7CK vs Am7T comparison group, and 611 up-regulated and 360 down-regulated genes in Am10CK vs Am10T comparison group. Venn analysis suggested that the number of specific DEGs of the aforementioned two comparison groups was 739 and 853, respectively, and 118 DEGs were shared. GO classification indicated that the up- and down-regulated genes in Am7CK vs Am7T were respectively annotated to 23 and 29 functional terms; among them, the top five terms of up-regulated genes were binding, catalytic activity, metabolic process, cellular process and single tissue process; while the top five terms of down-regulated genes were metabolic process, single tissue process, catalytic activity, cellular process and binding. In addition, the up- and down-regulated genes in Am10CK vs Am10T were respectively involved in 36 and 26 functional terms; among them, the top five terms of up-regulated genes were single process, binding, cellular process, catalytic activity and metabolic activity; while the top five terms of down-regulated genes were binding, cellular process, catalytic activity, metabolic process and single process. KEGG metabolic pathway enrichment analysis showed that the up- and down-regulated genes in Am7CK vs Am7T were respectively enriched in 38 and 33 pathways, and among them the top five enriched pathways of up-regulated genes were bile secretion, endoplasmic reticulum protein processing, ubiquitin-mediated proteolysis, PI3K-Akt signaling pathway and neurotrophic factor signaling pathway; while the top five enriched pathways of down-regulated genes were cytoplasmic DNA-sensing pathway, purine metabolism, pyrimidine metabolism, RNA polymerase and ribosome; three cellular immune pathways including ubiquitin-mediated proteolysis, and seven humoral pathways including PI3K-Akt signaling pathway were detected. Additionally, the up- and down-regulated genes in Am10CK vs Am10T were respectively associated with 54 and 43 pathways, and among them the top five enriched pathways of up-regulated genes were Hippo signaling pathway, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, ubiquitin-mediated proteolysis, and sphingolipid metabolism; while the top five enriched pathways of down-regulated genes were mRNA surveillance pathway, sphingolipid signaling pathway, fructose and mannose metabolism, galactose metabolism, and sphingolipid metabolism; seven cellular immune pathways including ubiquitin-mediated proteolysis, and two humoral pathways including NF-κB signaling pathway were found. The result of RT-qPCR verification demonstrated the expression trend of six randomly selected DEGs was consistent with that of the sequencing data, which confirmed the reliability of the transcriptome data. Further analysis noted that NF-κB signaling pathway was activated by N. ceranae in both 7- and 10-day-old workers’ midguts of A. m. ligustica, followed by immediate initiation of three antimicrobial peptides such as apidaecin, defensin-1 and hymenoptaecin, indicating their key importance in host defense against N. ceranae invasion.【Conclusion】 The immune responses of A. m. ligusitca worker’s midgut to N. ceranae were uncovered at transcriptomic level, and it was revealed that the host made both cellular and humoral immune responses at the early stage of N. ceranae stress. The host cellular immune responses may play a major role in resisting pathogen invasion. The host cellular immune continued to increase at the later stage of fungal stress, while the humoral immune drastically weakened. The ubiquitin mediated proteolysis and enriched DEGs, NF-κB signaling pathway and enriched DEGs, and antibacterial peptide-encoded genes such as apidaecin, defensin-1 and hymenoptaecin were likely to play pivotal roles in host immune response to N. ceranae stress.

Key words: Apis mellifera ligustica, Nosema ceranae, midgut, immune response, cellular immune, humoral immune

付中民,陈华枝,刘思亚,祝智威,范小雪,范元婵,万洁琦,张璐,熊翠玲,徐国钧,陈大福,郭睿. 意大利蜜蜂响应东方蜜蜂微孢子虫胁迫的免疫应答[J]. 中国农业科学, 2019, 52(17): 3069-3082.

FU ZhongMin,CHEN HuaZhi,LIU SiYa,ZHU ZhiWei,FAN XiaoXue,FAN YuanChan,WAN JieQi,ZHANG Lu,XIONG CuiLing,XU GuoJun,CHEN DaFu,GUO Rui. Immune Responses of Apis mellifera ligustia to Nosema ceranae Stress[J]. Scientia Agricultura Sinica, 2019, 52(17): 3069-3082.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.17.014

https://www.chinaagrisci.com/CN/Y2019/V52/I17/3069

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)
Actin-F	CACTCCTGCTATGTATGTCGC
Actin-R	GGCAAAGCGTATCCTTCA
A-F	CATTGAGGATGAGCCCAT
A-R	CACGCTATCGTCAACTTCC
B-F	AGCGATTGAAGATGTGCC
B-R	ACGACAGGTGAAACCGAA
C-F	CATTGAGGATGAGCCCAT
C-R	GGTTTACCGCCATTCACA
D-F	TTCAGAACTCACGAGGCTG
D-R	AACCACAATGGCACCAGTA
E-F	CCTATGACAGGAGGGATGGT
E-R	GCACTGCCAGGTCTTCTACT
F-F	AACGAACCTTTGTCGGGT
F-R	TCCATCTGAATGCTGCCA

通路名称 Pathway name	通路ID Pathway ID	上调基因 Up-regulated gene	下调基因 Down-regulated gene
泛素介导的蛋白水解 Ubiquitin mediated proteolysis	ko04120	2	0
细胞凋亡 Apoptosis	ko04210	1	0
溶酶体 Lysosome	ko04142	0	1

通路名称 Pathway name	通路ID Pathway ID	上调基因 Up-regulated gene	下调基因 Down-regulated gene
PI3K-Akt信号通路 PI3K-Akt signaling pathway	ko04151	2	1
趋化因子信号通路 Chemokine signaling pathway	ko04062	1	0
NF-κB信号通路 NF-κB signaling pathway	ko04064	1	0
cAMP信号通路 cAMP signaling pathway	ko04024	0	1
Ras信号通路 Ras signaling pathway	ko04014	0	1
MAPK信号通路-酵母 MAPK signaling pathway - yeast	ko04011	0	1
FoxO信号通路 FoxO signaling pathway	ko04068	0	1

通路名称 Pathway name	通路ID Pathway ID	上调基因 Up-regulated gene	下调基因 Down-regulated gene
泛素介导的蛋白水解 Ubiquitin mediated proteolysis	ko04120	2	0
P450对外源物质代谢 Metabolism of xenobiotics by cytochrome P450	ko00980	2	0
药物代谢-细胞色素P450 Drug metabolism - cytochrome P450	ko00982	2	0
细胞凋亡 Apoptosis	ko04210	1	0
Fc-γ-R介导的吞噬作用 Fc-γ-R-mediated phagocytosis	ko04666	1	0
溶酶体 Lysosome	ko04142	1	0
内吞作用 Endocytosis	ko04144	1	1

通路名称 Pathway name	通路ID Pathway ID	上调基因 Up-regulated gene	下调基因 Down-regulated gene
NF-κB信号通路 NF-κB signaling pathway	ko04064	1	0
PI3K-Akt信号通路 PI3K-Akt signaling pathway	ko04151	0	1