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

中国农业科学 ›› 2020, Vol. 53 ›› Issue (17): 3606-3619.doi: 10.3864/j.issn.0578-1752.2020.17.017

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

蜜蜂球囊菌菌丝和孢子中微小RNA及其靶mRNA的比较分析

陈华枝(),祝智威(),蒋海宾,王杰,范元婵,范小雪,万洁琦,卢家轩,熊翠玲,郑燕珍,付中民,陈大福,郭睿()

福建农林大学动物科学学院（蜂学学院）,福州 350002

收稿日期:2019-12-25 接受日期:2020-02-04 出版日期:2020-09-01 发布日期:2020-09-11
通讯作者: 郭睿
作者简介:陈华枝,E-mail：CHZ0720@outlook.com。|祝智威,E-mail：zzw15235470398@163.com。
基金资助:
国家自然科学基金(31702190);国家现代农业产业技术体系建设专项(CARS-44-KXJ7);福建省自然科学基金(2018J05042);福建省教育厅中青年教师教育科研项目(JAT170158);福建农林大学杰出青年科研人才计划(xjq201814);福建农林大学科技创新专项(CXZX2017342, CXZX2017343);福建农林大学优秀硕士学位论文资助基金(陈华枝)

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

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()

College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002

Received:2019-12-25 Accepted:2020-02-04 Online:2020-09-01 Published:2020-09-11
Contact: Rui GUO

摘要/Abstract

摘要：

【目的】蜜蜂球囊菌(Ascosphaera apis,球囊菌)专性侵染蜜蜂幼虫而导致白垩病。本研究旨在通过small RNA-seq（sRNA-seq）技术和生物信息学方法对球囊菌纯化菌丝（AaM）和纯化孢子（AaS）进行深度测序和比较分析,明确球囊菌菌丝miRNA和孢子miRNA的数量、结构和表达谱差异,并揭示菌丝和孢子共有miRNA、特有miRNA和差异表达miRNA（differentially expressed miRNA,DEmiRNA）及其靶mRNA与球囊菌菌丝和孢子生长、发育和病原致病性的潜在关系。【方法】实验室条件下获得纯培养的球囊菌,利用sRNA-seq技术对AaM和AaS分别进行测序,通过对原始读段（raw reads）进行过滤和质控获得有效标签序列（clean tags）。通过Venn分析筛选菌丝和孢子共有miRNA和特有miRNA。根据P≤0.05且|log₂fold change|≥1的标准筛选AaM vs AaS的DEmiRNA。对上述共有miRNA、特有miRNA和DEmiRNA的靶mRNA进行预测,并对靶mRNA进行GO及KEGG数据库注释。根据靶向结合关系构建DEmiRNA和靶mRNA的调控网络。利用RT-qPCR验证测序数据的可靠性。【结果】AaM和AaS中分别得到12 982 320和12 708 832条raw reads,经过滤和质控分别得到10 800 101和9 888 848条clean tags。AaM中miRNA的长度介于18—26 nt,AaS中miRNA的长度介于18—24 nt,分布miRNA数量最多的长度均为18 nt,AaM和AaS中首位碱基为U的miRNA数量最多。AaM和AaS中表达量最高的miRNA均为miR6478-x、miR10516-x和miR482-x。菌丝和孢子共有miRNA靶向结合5 946个mRNA,二者特有miRNA分别靶向结合6 141和6 346个mRNA。共有miRNA的靶mRNA主要参与代谢进程、细胞进程和催化活性等42个功能条目,以及翻译、碳水化合物代谢和能量代谢等120条通路。AaM vs AaS比较组包含93个DEmiRNA,可靶向结合6 090个mRNA,这些靶mRNA可注释到38个功能条目和120条通路。DEmiRNA与靶mRNA之间形成较为复杂的调控网络,miR-4968-y位于调控网络的中心且能够靶向结合多达118个mRNA。RT-qPCR结果显示5个DEmiRNA的表达趋势与测序数据一致,证实了本研究中测序数据的可靠性。【结论】球囊菌菌丝和孢子中的miRNA具有类似的结构特征,但表达谱表现出明显差异;菌丝和孢子可能通过特异性表达和差异表达部分miRNA对其生长、发育和生殖进行调控。

关键词: 蜜蜂球囊菌, 菌丝, 孢子, 微小RNA, 信使RNA, 靶向结合

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

陈华枝,祝智威,蒋海宾,王杰,范元婵,范小雪,万洁琦,卢家轩,熊翠玲,郑燕珍,付中民,陈大福,郭睿. 蜜蜂球囊菌菌丝和孢子中微小RNA及其靶mRNA的比较分析[J]. 中国农业科学, 2020, 53(17): 3606-3619.

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

蜜蜂球囊菌菌丝和孢子中微小RNA及其靶mRNA的比较分析

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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