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

doi:10.3864/j.issn.0578-1752.2019.01.015

摘要/Abstract

摘要：

【目的】微小RNA（microRNA,miRNA）是一类重要的基因表达调控因子,可影响宿主与病原间的互作过程。蜜蜂球囊菌（Ascosphaera apis）是一种特异性侵染蜜蜂幼虫的致死性真菌病原。本研究旨在对意大利蜜蜂（Apis mellifera ligustica,简称意蜂）幼虫肠道在球囊菌胁迫前期的差异表达miRNA（differentially expressed miRNA,DEmiRNA）及其靶基因进行深入分析,在miRNA组学水平探究意蜂幼虫在球囊菌侵染前期的胁迫应答,并通过构建显著DEmiRNA的调控网络筛选出与宿主应答相关的关键miRNA。【方法】利用small RNA-seq（sRNA-seq）技术对正常及球囊菌侵染的意蜂4日龄幼虫肠道（AmCK和AmT）进行高通量测序,首先对原始数据进行质控和评估,随后将过滤后的数据与西方蜜蜂（Apis mellifera）参考基因组进行比对;将比对上的序列标签（tags）注释到miRBase数据库,得出已知miRNA的表达量;通过TPM（tags per million）算法对各样本中miRNA的表达量进行归一化处理,以|log₂ fold change|≥1且P≤0.05作为标准筛选得到显著DEmiRNA;利用TargetFinder 软件预测显著DEmiRNA的靶基因,并对其进行GO和KEGG代谢通路（pathway）富集分析。利用Cytoscape软件对miRNA-mRNA调控网络进行可视化。最后,利用茎环反转录PCR（Stem-loop RT-PCR）和荧光定量PCR（qPCR）验证测序数据的可靠性。【结果】 AmCK和AmT样品的测序分别得到13 553 302和10 777 534条原始读段（raw reads）,经严格过滤后得到的有效读段（clean reads）数分别为13 186 921和10 480 913条。各样品的生物学重复间的Pearson相关性系数分别在0.9822和0.9508以上。共有10个显著DEmiRNA,包括4个上调miRNA和6个下调miRNA。显著DEmiRNA在AmT的整体表达水平低于AmCK。10个显著DEmiRNA可靶向结合3 788个靶基因,其中上调miRNA的1 240个靶基因可注释到GO数据库中的39个GO条目,主要富集在结合、细胞进程、代谢进程和应激反应等;下调miRNA的749个靶基因可注释到34个GO条目,主要富集在细胞进程、结合、代谢进程和应激反应等。KEGG数据库注释结果显示,上调miRNA和下调miRNA的靶基因分别注释到95和66条代谢通路,富集基因数最多的分别是Wnt信号通路、Hippo信号通路、光传导以及内吞作用、磷脂酰肌醇信号系统、嘌呤代谢。对于上调和下调miRNA,分别有31和52个靶基因注释到内吞作用,15和7个靶基因注释到泛素介导的蛋白水解,11和5个靶基因注释到Jak-STAT信号通路,1和3个靶基因注释到MAPK信号通路。显著DEmiRNA与靶mRNA之间形成复杂的调控网络,7个显著DEmiRNA靶向结合96个与Wnt信号通路相关的mRNA,8个显著DEmiRNA靶向结合55个与内吞作用相关的mRNA。Stem-loop RT-PCR和qPCR结果验证了测序数据的可靠性。【结论】对意蜂幼虫肠道在球囊菌侵染前期的DEmiRNA及其靶基因进行预测和分析,并构建和分析了DEmiRNA-mRNA调控网络,研究结果提供了宿主miRNA的表达谱和差异表达信息,揭示了DEmiRNA通过调控细胞生命活动、新陈代谢以及部分细胞和体液免疫等生物学过程参与宿主的胁迫应答。miR-4331-y、miR-4968-y、miR-8440-y、novel-m0023-5p和novel-m0025-3p共同参与了宿主的Wnt信号通路和内吞作用的调控,可作为白垩病治疗的潜在分子靶标。

关键词: 意大利蜜蜂, 幼虫肠道, 发育, 差异表达微小RNA, 调控网络

Abstract:

【Objective】MicroRNA (miRNA) is a kind of key gene expression regulator, which can affect the interactions between host and pathogen. Ascosphaera apis is a lethal fungal pathogen that specifically infects honeybee larvae. The objective of this study is to analyze the differentially expressed miRNAs (DEmiRNAs) and their target genes in the Apis mellifera ligustica larval gut during the early infection stage of A. apis, reveal DEmiRNA’ roles in the stress responses of host at the miRNA omics level, and to screen the key miRNAs related to host response by constructing regulation networks of significant DEmiRNAs. 【Method】Normal and A. apis-infected 4-day-old larval gut of A. m. ligustica (AmCK and AmT) were deep-sequenced using small RNA-seq (sRNA-seq) technology, followed by quality-control of raw data and then mapping of the filtered data with the reference genome of Apis mellifera. The mapped tags were compared to the miRBase database to identify the expression of known miRNAs. The expression of miRNAs in each sample was normalized by TPM (tags per million) algorithm and significant DEmiRNAs were gained according to the standard |log₂fold change|≥1 and P≤0.05. Target genes of significant DEmiRNAs were predicted utilizing TargetFinder, and then annotated to the GO and KEGG databases. Cytoscape was used to visualize the regulation networks between significant DEmiRNAs and target mRNAs. Finally, Stem-loop RT-PCR and qPCR were conducted to verify the reliability of the sequencing data.【Result】sRNA-seq of AmCK and AmT produced 13 553 302 and 10 777 534 raw reads, and after strict filtration, 13 186 921 and 10 480 913 clean reads were obtained, respectively. The Pearson correlation coefficients among different biological replicates in each sample were above 0.9822 and 0.9508. There were 10 significant DEmiRNAs including 4 up-regulated miRNAs and 6 down-regulated miRNAs, and the overall expression level of DEmiRNAs in AmT was lower than that in AmCK. In total, 10 significant DEmiRNAs could link 3 788 target genes. The 1 240 target genes of up-regulated miRNAs could be annotated to 39 GO terms, and the mostly enriched terms were binding, cellular processes, metabolic processes, and response to stimulus. The 749 target genes of down-regulated miRNAs could be annotated to 34 GO terms, and the mostly enriched terms were cellular processes, binding, metabolic processes, and response to stimulus. The result of KEGG database annotation suggested that the target genes of up- and down-regulated miRNAs were respectively annotated in 95 and 66 pathways, the most abundant pathways were Wnt signaling pathway, Hippo signaling pathway, phototransduction and endocytosis, phosphatidylinositol signaling system, as well as purine metabolism. For up- and down-regulated miRNAs, there were 31 and 52 target genes could be annotated to endocytosis, 15 and 7 target genes could be annotated to ubiquitin-mediated proteolysis, 11 and 5 target genes could be annotated to Jak-STAT signaling pathway, 1 and 3 target genes could be annotated to the MAPK signaling pathway, respectively. Complex regulation networks existed between significant DEmiRNAs and their target mRNAs, among them 7 significant DEmiRNAs targeted 96 mRNAs associated with Wnt signaling pathway, and 8 significant DEmiRNAs targeted 55 mRNAs involved in endocytosis. Finally, the results of Stem-loop RT-PCR and qPCR verified the reliability of the sequencing data.【Conclusion】A. m. ligustica larval gut’s DEmiRNAs and their target genes during the early infection stage of A. apis were predicted and analyzed. DEmiRNA-mRNA regulation networks in the host were constructed and investigated. The results provide the expression profile and differential expression information of host miRNAs, and reveal that these DEmiRNAs likely participate in the stress responses of host via regulating biological processes such as cellular activity, metabolism, and immune defense. miR-4331-y, miR-4968-y, miR-8440-y, novel-m0023-5p and novel-m0025-3p jointly regulate Wnt signaling pathway and endocytosis of host and can be used as potential molecular targets for chalkbrood control.

Key words: Apis mellifera ligustica, larval gut, development, differentially expressed microRNA, regulation network

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[J]. 中国农业科学, 2019, 52(1): 166-180.

GUO Rui,DU Yu,TONG XinYu,XIONG CuiLing,ZHENG YanZhen,XU GuoJun,WANG HaiPeng,GENG SiHai,ZHOU DingDing,GUO YiLong,WU SuZhen,CHEN DaFu. Differentially Expressed MicroRNAs and Their Regulation Networks in Apis mellifera ligustica Larval Gut During the Early Stage of Ascosphaera apis Infection[J]. Scientia Agricultura Sinica, 2019, 52(1): 166-180.

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引物名称 Primer name	引物序列 Primer sequence
LOOP-miR-3793-x	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTGGCCAGG
LOOP-ame-miR-6000a-5p	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATAGAGAC
LOOP-novel-m0031-3p	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCCTGCTT
LOOP-novel-m0034-5p	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATATCACA
F-miR-3793-x	ACACTCCAGCTGGGAGCGTGTTTTC
F-ame-miR-6000a-5p	ACACTCCAGCTGGGCAGCAGCAGCAG
F-novel-m0031-3p	GCATCCTCTTGAAT
F-novel-m0034-5p	CAGGTAACTACTGC
R	CTCAACTGGTGTCGTGGA
U6-F	GTTAGGCTTTGACGATTTCG
U6-R	GGCATTTCTCCACCAGGTA

样品Sample	原始读段Raw reads	有效读段Clean reads
AmCK-1	15146178	14736731 (97.30%)
AmCK-2	13310167	12954535 (97.33%)
AmCK-3	12203560	11869496 (97.26%)
AmT-1	10741447	10453795 (97.32%)
AmT-2	12035887	11694678 (97.17%)
AmT-3	9555268	9294267 (97.27%)

差异表达miRNA ID DEmiRNA ID	AmCK中的表达量 Expression in AmCK	AmT中的表达量 Expression in AmT	以2为底miRNA的相对变化倍数的对数值 log₂fold change	靶基因数 Number of target genes
miR-7085-x	0.01	8.40	9.71	129
miR-8440-y	0.01	5.29	9.05	2265
novel-m0023-5p	0.01	1.79	7.48	334
miR-3793-x	33.72	89.76	1.41	398
novel-m0034-3p	4.06	1.95	-1.05	768
miR-4331-y	74.07	27.70	-1.42	77
ame-miR-6000a-5p	19.06	4.14	-2.20	77
miR-971-y	3.25	0.31	-3.37	100
novel-m0025-3p	0.88	0.01	-6.46	294
miR-4968-y	11.61	0.01	-10.18	279