蜜蜂球囊菌菌丝和孢子中微小RNA及其靶mRNA的比较分析

doi:10.3864/j.issn.0578-1752.2020.17.017

Abstract

Abstract: 【Objective】Ascosphaera apis exclusively infects honeybee larvae, resulting in chalkbrood disease. The objective of this study is to clarify the differences of number, structure and expression pattern of miRNAs between A. apis mycelium and spore based on deep sequencing and comparative analysis of purified mycelia (AaM) and spores (AaS) using small RNA-seq (sRNA-seq) and bioinformatics, and reveal the potential relationship between common miRNAs, specific miRNAs, differentially expressed miRNAs (DEmiRNAs) and their target mRNAs and the growth and development of mycelium and spore as well as pathogenesis of A. apis.【Method】The pure culture of A. apis was gained under lab condition. AaM and AaS were respectively sequenced using sRNA-seq technology. Clean tags were obtained after filtration and quality control of raw reads. Common miRNAs and specific miRNAs in AaM and AaS were screened out using Venn analysis. DEmiRNAs in the AaM vs AaS comparison group were filtered out following the criteria of P≤0.05 and |log2 fold change|≥1. Target mRNAs of common miRNAs, specific miRNAs and DEmiRNAs were predicted using related bioinformatic software. Target mRNAs mentioned above were respectively annotated to GO database and KEGG database. The regulatory network of DEmiRNAs and target mRNAs was constructed on basis of target binding relationship, followed by visualization with Cytoscape. RT-qPCR was conducted to verify the reliability of the sequencing data.【Result】In total, 12 982 320 and 12 708 832 raw reads were produced from AaM and AaS, and after strict quality control, 10 800 101 and 9 888 848 clean tags were gained, respectively. The length of specific miRNAs in AaM was distributed among 18-26 nt, while that in AaS was distributed among 18-24 nt. Additionally, most of the miRNAs were distributed in 18 nt. MiRNAs with the first base U in both AaM and AaS were the most abundant. MiRNAs with the highest expression levels in both AaM and AaS were miR6478-x, miR10516-x and miR482-x. These common miRNAs could target 5 946 mRNAs, while specific miRNAs in AaM and AaS could bind to 6 141 and 6 346 mRNAs, respectively. Targets of common miRNAs were annotated to 42 functional terms such as metabolism process, cellular process and catalytic activity, and 120 pathways including translation, carbohydrate metabolism and energy metabolism. In addition, a total of 93 DEmiRNAs were identified in AaM vs AaS comparison group, targeting 6 090 mRNAs annotated to 38 functional terms and 120 pathways. Moreover, complicated regulatory networks were formed between DEmiRNAs and target mRNAs, with miR-4968-y located in the center and linked to as many as 118 mRNAs. RT-qPCR result demonstrated the expression trend of five DEmiRNAs was consistent with that in the sequencing result, confirming the reliability of our sequencing data.【Conclusion】The structures of miRNAs in A. apis mycelium and spore were similar, whereas their expression patterns were obviously different; mycelium and spore may specifically and differentially express part of miRNAs to regulate their growth, development and reproduction.

Key words: Ascosphaera apis, mycelium, spore, miRNA, mRNA, target binding

CHEN HuaZhi,ZHU ZhiWei,JIANG HaiBin,WANG Jie,FAN YuanChan,FAN XiaoXue,WAN JieQi,LU JiaXuan,XIONG CuiLing,ZHENG YanZhen,FU ZhongMin,CHEN DaFu,GUO Rui. Comparative Analysis of MicroRNAs and Corresponding Target mRNAs in Ascosphaera apis Mycelium and Spore[J].Scientia Agricultura Sinica, 2020, 53(17): 3606-3619.

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引物名称 Primer name	引物序列Primer sequence (5′-3′)
Loop-miR5658-x	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCATCATC
Loop-miR-10285-y	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACAATTGG
Loop-miR-3245-y	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCCCGGAC
Loop-miR4404-x	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCCAGTCT
Loop-miR-9-z	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCATACAG
miR5658-x-F	ACACTCCAGCTGGGCGATGATGAT
miR-10285-y-F	ACACTCCAGCTGGGTCATTTGTGGT
miR-3245-y-F	ACACTCCAGCTGGGCTTGGGAGAG
miR4404-x-F	ACACTCCAGCTGGGAATACGTAGA
miR-9-z-F	ACACTCCAGCTGGGTCTTTGGTTATCTAG
Universal-R	CTCAACTGGTGTCGTGGA
actin-F	CAGGAAAGGCTATGTTCGC
actin-R	ATTACCGAGGAGCAAGACG

样品 Sample	原始读段 Raw reads	有效标签序列 Clean tags	比对上参考基因组的clean tags Clean tags mapped to reference genome
AaM	12982320	10800101 (84.34%)	8703440 (80.59%)
AaS	12708832	9888848 (78.83%)	7080369 (71.60%)

miRNA ID	miRNA序列 miRNA sequence	TPM值 TPM value
miR6478-x	GCGACTTTAGCTCAGTTGG	294585.5787
miR10516-x	GATCCTCTGCAGACGACTGA	200923.7875
miR482-x	TTGGAGTGGGTGGGTTGGGAA	99563.767
miR-8440-y	GTTCGTTTCTGGGTCAGG	84423.9158
miR5658-x	CGATGATGATGATGATGA	30792.9176
miR-11987-x	AGGAAACTCTGGTGGAGGT	27713.6259
miR-10285-y	TCATTTGTGGTCCAATTGT	11547.3441
miR-3245-y	CTTGGGAGAGGTCCGGGG	10264.3059
miR-1332-y	CAGTTGGTTAGAGCTGGT	9494.4829

miRNA ID	miRNA序列 miRNA sequence	TPM值 TPM value
miR6478-x	GCGACTTTAGCTCAGTTGG	209814.8692
miR482-x	TTGGAGTGGGTGGGTTGGGAA	71995.2983
miR10516-x	GATCCTCTGCAGACGACTGA	71407.5815
miR-21-x	TAGCTTATCAGACTGATGTTGA	32324.4196
miR-8440-y	GTTCGTTTCTGGGTCAGG	31736.7029
miR-143-y	TGAGATGAAGCACTGTAGCTCT	29973.5527
miR-11987-x	AGGAAACTCTGGTGGAGGT	26741.1108
let-7-x	TGAGGTAGTAGGTTGTATAGTT	19688.5101
novel-m0040-3p	TCTTGAACTGAGAGATGGGGC	16456.0682
miR159-y	TCTTGGGGTGAAGGGCGG	15574.4931

差异表达miRNA ID DEmiRNA ID	AaM的TPM值 TPM value in AaM	AaS的TPM值 TPM value in AaS	Log₂差异倍数 Log₂FC	P 值 P value
上调miRNA Up-regulated miRNAs
novel-m0040-3p	0.01	16456.0682	20.65019	5.98E-17
novel-m0016-3p	0.01	14399.0597	20.45754	3.77E-15
miR319-y	0.01	12342.0511	20.23515	4.75E-13
novel-m0010-3p	0.01	8521.8924	19.70081	3.80E-09
novel-m0028-3p	0.01	7346.459	19.48669	6.05E-08
miR-1546-x	0.01	5583.3088	19.09076	3.85E-06
miR-6882-x	0.01	5289.4505	19.01276	7.69E-06
novel-m0001-3p	0.01	4995.5921	18.9303	1.54E-05
novel-m0017-5p	0.01	4701.7338	18.84283	3.07E-05
miR-992-x	0.01	4701.7338	18.84283	3.07E-05
下调miRNA Down-regulated miRNAs
miR-4028-y	8211.4447	0.01	-19.6472765	4.77E-10
miR-4171-x	3079.2918	0.01	-18.2322391	0.00049
miR7787-y	2822.6841	0.01	-18.1067082	0.000979
miR-92-x	2566.0765	0.01	-17.9692047	0.0020
novel-m0011-3p	2309.4688	0.01	-17.8172015	0.0040
miR5782-y	2052.861	0.01	-17.6473	0.0078
novel-m0013-5p	2052.861	0.01	-17.6473	0.0078
novel-m0031-3p	2052.861	0.01	-17.6473	0.0078
novel-m0036-5p	2052.861	0.01	-17.6473	0.0078
miR-3533-y	1796.254	0.01	-17.4546	0.0156
novel-m0003-5p	1796.254	0.01	-17.4546	0.0156
novel-m0006-5p	1796.254	0.01	-17.4546	0.0156

Comparative Analysis of MicroRNAs and Corresponding Target mRNAs in Ascosphaera apis Mycelium and Spore

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通路名称 Pathway name	通路 ID Pathway ID	靶mRNA数 Number of target mRNAs	P 值 P value
新陈代谢通路Metabolic pathway	ko01100	652	0.174
次级代谢物的生物合成Biosynthesis of secondary metabolites	ko01110	285	5.63E-05
抗生素的生物合成Biosynthesis of antibiotics	ko01130	212	0.002
微生物在不同环境中的代谢Microbial metabolism in diverse environments	ko01120	172	0.001
氨基酸的生物合成Biosynthesis of amino acids	ko01230	115	0.012
碳代谢Carbon metabolism	ko01200	101	0.025
嘌呤代谢Purine metabolism	ko00230	81	0.916
核糖体Ribosome	ko03010	78	1.000
剪接体Spliceosome	ko03040	77	0.861
RNA转运RNA transport	ko03013	76	0.936

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