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

Abstract

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

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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References 46

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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

Differentially Expressed MicroRNAs and Their Regulation Networks in Apis mellifera ligustica Larval Gut During the Early Stage of Ascosphaera apis Infection

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