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

doi:10.3864/j.issn.0578-1752.2018.23.015

Abstract

Abstract:

【Objective】Circular RNA (circRNA) plays a primary role in alternative splicing, transcription regulation and expression regulation of parental gene. The objective of this study is to investigate the profile expression of circRNAs and differentially expressed circRNAs (DEcircRNAs) during the developmental process of the midguts of Apis mellifera ligustica workers, and to explore the role of DEcircRNAs in the development of midgut at the transcriptional level. 【Method】 On basis of the whole transcriptome data from 7- and 10-day-old worker’s midguts of A. m. ligustica (Am7 and Am10), find_circ software was used to predict circRNAs based on the filtered sequencing data. The circRNA expression level was normalized by RPM algorithm. Differential expression analysis for circRNAs was conducted via DEGseq software following standards fold change≥2.0, P<0.05 and false discovery rate (FDR) <0.05. Source genes of DEcircRNAs were annotated to GO and KEGG databases to gain function and pathway annotations by using BLAST. DEcircRNAs-miRNAs and DEcircRNAs-miRNAs-mRNAs networks were predicted with TargetFinder software and visualized using Cytoscape v.3.2.1 software. RT-qPCR was conducted to verify the reliability of sequencing data.【Result】 On average, 19 616 356 anchors reads were obtained from each A. m. ligustica worker’s midgut sample. Pearson correlations between different biological repeats within Am7 and Am10 groups were ≥0.950. In total, 256 DEcircRNAs including 105 up-regulated circRNAs and 151 down-regulated circRNAs were predicted. Novel_circ_009675 and novel_circ_013879 were highly expressed in Am7 and Am10, respectively. Source genes of DEcircRNAs could be annotated to 32 GO terms including binding, single-organism process and cellular process, among them 35, 35 and 7 source genes were involved in catalytic activity, metabolic process and stress response. Additionally, these source genes could be annotated to 35 KEGG pathways, in which 5, 5 and 4 source genes were associated with Hippo signaling pathway, endocytosis and phagosome, respectively; further investigation showed that 1, 2 and 2 source genes could be annotated to material metabolisms such as phosphoinositol metabolism, starch and sucrose metabolism and galactose metabolism; 5, 4, 3, 1 and 1 source genes could be annotated to immune signaling pathways including endocytosis, phagosome, lysosome, ubiquitin-mediated proteolysis and MAPK signaling pathway, respectively. These results suggested that the corresponding DEcircRNA was involved in the development, metabolism and immune defense of the midgut of A. m. ligustica. DEcircRNA-miRNA regulation network analysis showed that 141 DEcircRNAs could link to 107 miRNAs, most of these DEcircRNAs could only bind to 1-2 miRNAs, but novel_circ_011577 and novel_circ_010719 could respectively bind to 32 and 28 miRNAs. In addition, the number of DEcircRNAs combined with mir-136-y, ame-miR-6001-3p and mir-136-y was the highest (15, 14 and 14, respectively), which indicated that the corresponding DEcircRNA could play roles during the developmental process of A. m. ligustica worker’s midgut as competing endogenous RNAs. Furthermore, DEcircRNAs-ame-miR-6001-3p-mRNA network was constructed and analyzed, and the result indicated that 14 DEcircRNAs could jointly link to ame-miR-6001-3p, implying they were likely to indirectly regulate division and differentiation of stem cells in A. m. ligustica worker’s midgut via regulation of ame-miR-6001-3p. Six DEcircRNAs were randomly selected for RT-qPCR assay, the result showed the alteration trend of expression levels of 5 DEcircRNAs was consistent with that of the sequencing data, which proved the reliability of trancriptome data.【Conclusion】Through the deep investigation of DEcircRNAs during the developmental process of A. m. ligustica worker’s midgut, the expression profile and differential expression information of circRNAs in the development of midgut of worker bee were provided, and the role of DEcircRNAs in the development of midgut was revealed. It provides a basis for the screening and functional study of key circRNAs associated with the development of the midgut.

Key words: Apis mellifera ligustica, midgut, circRNAs, regulation network, development

GUO Rui,CHEN HuaZhi,XIONG CuiLing,ZHENG YanZhen,FU ZhongMin,XU GuoJun,DU Yu,WANG HaiPeng,GENG SiHai,ZHOU DingDing,LIU SiYa,CHEN DaFu. Analysis of Differentially Expressed Circular RNAs and Their Regulation Networks During the Developmental Process of Apis mellifera ligustica Worker’s Midgut[J].Scientia Agricultura Sinica, 2018, 51(23): 4575-4590.

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References 56

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引物名称Primer name	引物序列Primer sequence
Novel_circ_005547-F	TCTGCTACTCAAATGGAGGG
Novel_circ_005547-R	CCCACTGTCTCTCTTCTAAGGA
Novel_circ_014049-F	GGAAGGAAGGAAGTAGCGA
Novel_circ_014049-R	CACGAACACCACCCAATA
Novel_circ_002507-F	ATTTCCTTGGGCATAGCC
Novel_circ_002507-R	CTCGGTCAAACCATACACC
Novel_circ_012440-F	AGTCTGTTCGGTAATCCCG
Novel_circ_012440-R	CTCACCTGATACTTCACCTTTG
Novel_circ_001915-F	CATCATCTCCGAAACCGA
Novel_circ_001915-R	TTGAGGTGGCTGACTTGA
Actin-F	CACTCCTGCTATGTATGTCGC
Actin-R	GGCAAAGCGTATCCTTCA

样品 Sample	短序列读段 Anchors reads	比对上的短序列读段 Mapped anchors reads	比对率 Mapping ratio (%)
Am7-1	236400908	19006601	8.04
Am7-2	168420472	19873468	11.80
Am7-3	136779122	17502228	12.80
Am10-1	195020440	22441139	11.51
Am10-2	184091234	21109367	11.47
Am10-3	168790774	17765330	10.53

CircRNA名称 Name of circRNAs	RPM值 RPM value	来源基因ID ID of source gene	长度 Length (bp)	环化类型 Type of circularization
novel_circ_003183	45581.85	410805	552	annot_exons
novel_circ_010717	33244.11	413053	956	annot_exons
novel_circ_012530	21253.62	725393	1190	one_exon
novel_circ_000476	15898.09	552294	1742	annot_exons
novel_circ_011750	14352.44	410379	613	antisense
novel_circ_011749	14094.35	410379	613	antisense
novel_circ_000896	12421	100576586	1362	annot_exons
novel_circ_006484	9348.5	413332	419	annot_exons
novel_circ_009675	7112.647	408996	404	annot_exons
novel_circ_012115	6698.064	724592	727	annot_exons

CircRNA名称 Name of circRNAs	RPM值 RPM value	来源基因ID ID of source gene	长度 Length (bp)	环化类型 Type of circularization
novel_circ_003183	50507.89	410805	552	annot_exons
novel_circ_010717	39732.13	413053	956	annot_exons
novel_circ_012530	23025.9	725393	1190	one_exon
novel_circ_000896	17755.41	100576586	1362	annot_exons
novel_circ_000476	17621.29	552294	1742	annot_exons
novel_circ_011750	13241.09	410379	613	antisense
novel_circ_011749	12729.28	410379	613	antisense
novel_circ_006484	9038.38	413332	419	annot_exons
novel_circ_013879	7982.833	408521	1219	exon_intron
novel_circ_012115	7316.428	724592	727	annot_exons

Analysis of Differentially Expressed Circular RNAs and Their Regulation Networks During the Developmental Process of Apis mellifera ligustica Worker’s Midgut

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