Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (1): 208-218.doi: 10.3864/j.issn.0578-1752.2022.01.017

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Identification and Analysis of MicroRNAs in the Larval Gut of Apis cerana cerana

FENG RuiRong1(),FU ZhongMin1,2(),DU Yu1,ZHANG WenDe1,FAN XiaoXue1,WANG HaiPeng1,WAN JieQi1,ZHOU ZiYu1,KANG YuXin1,CHEN DaFu1,2,GUO Rui1,2(),SHI PeiYing1()   

  1. 1College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002
    2Apitherapy Research Institute, Fujian Agriculture and Forestry University, Fuzhou 350002
  • Received:2021-04-28 Accepted:2021-06-07 Online:2022-01-01 Published:2022-01-07
  • Contact: Rui GUO,PeiYing SHI E-mail:frr18705911634@163.com;369699776@qq.com;ruiguo@fafu.edu.cn;peiyshi@126.com

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Abstract:

【Objective】 In this study, transcriptome-wide identification and analysis of miRNAs in the larval guts of Apis cerana cerana was conducted using a combination of small RNA-seq (sRNA-seq) technology and bioinformatic method, aiming to enrich the information of A. c. cerana miRNAs and offer a basis for further investigation of miRNA-regulated molecular mechanism underlying A. c. cerana larval gut development.【Method】 Gut samples of A. c. cerana 4-, 5-, and 6-day-old larvae (Ac1, Ac2, and Ac3 ) were sequenced using sRNA-seq technology, and clean tags were obtained after quality control. By using Blast tool, clean tags were continuously mapped to Apis cerana genome and miRBase database to identify known miRNAs and novel miRNAs. TPM method was used to perform normalization of miRNAs’ expression. The ratio of sRNAs, length distribution of miRNAs and first base bias were calculated with GraphPad Prism 7 software. Using related software, target mRNAs of above-mentioned miRNAs were predicted followed by GO and KEGG database annotation. Further, regulatory networks between genes associated with development and immune-related pathways and corresponding target miRNAs were constructed and analyzed, followed by visualization of regulatory networks with Cytoscape software. The authenticity of miRNA expression and sequence was verified by using Stem-loop RT-PCR, molecular cloning and Sanger sequencing.【Result】 In total, 371 known miRNAs and 64 novel ones of A. c. cerana were identified; their length was distributed among 18-25 nt, and the first base had an U bias. The aforementioned miRNAs could target 14 750 mRNAs, involving 2 270 GO terms such as ion binding, metal ion binding, membrane, membrane part and single-organism process, as well as 332 KEGG pathways including endocytosis, apoptosis, mTOR signaling pathway, RNA transport and insect hormone biosynthesis. Further investigation suggested that 156 miRNAs could target 67 genes relative to development-associated pathways such as Wnt, Hippo, Notch and mTOR signaling pathways, while 145 miRNAs could target 21 genes relevant to immune-associated pathways such as Toll, Imd/JNK, Jak-STAT signaling pathways and antimicrobial effectors. Stem-loop RT-PCR result indicated that specific fragments with expected sizes could be amplified from miR-8-y, miR-9-z, miR-14-y, miR-281-y, miR-283-x and miR-306-x; Sanger sequencing result demonstrated that sequences of above-mentioned six miRNAs were in accordance with those in deep sequencing result.【Conclusion】 Our findings provide number, structural characteristics and expression profile of A. c. cerana miRNAs, and unraveled that miRNAs in A. c. cerana larval gut potentially regulate a lot of life processes and cellular activities, part of miRNAs can participate in regulation of development-related and immune-related pathways by targeting corresponding mRNAs.

Key words: Apis cerana, Apis cerana cerana, larva, gut, microRNA, regulatory network

Table 1

An overview of sRNA-seq dataset"

样品
Sample		 高质量读段
High quality reads		 有效序列标签
Clean tags		 占比
Ratio (%)
Ac1-1 11231070 9761109 86.91
Ac1-2 11994516 10566352 88.09
Ac1-3 10594332 9048317 85.41
Ac2-1 13057999 10932504 83.72
Ac2-2 10718330 8834247 82.42
Ac2-3 10273562 8440841 82.16
Ac3-1 10611126 8828377 83.20
Ac3-2 9932874 8401872 84.59
Ac3-3 12822276 10953694 85.43

Fig. 1

Structural characteristics of miRNAs in A. c. cerana larval guts"

Fig. 2

Confirmation of miRNAs in A. c. cerana larval gut using Stem-loop RT-PCR and Sanger sequencing"

Fig. 3

Loop graph showing GO terms enriched by target mRNAs of miRNAs in the larval gut of A. c. cerana"

Fig. 4

Loop graph showing KEGG pathways enriched by target mRNAs of miRNAs in the larval gut of A. c. cerana"

Fig. 5

Regulation network between miRNAs and development-associated target genes in A. c. cerana larval gut"

Fig. 6

Regulation network between miRNAs and immune-associated target genes in A. c. cerana larval gut"

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