摘要
【目的】利用small RNA-seq(sRNA-seq)技术和生物信息学方法对中华蜜蜂(Apis cerana cerana,简称中蜂)幼虫肠道的微小RNA(microRNA,miRNA)进行全转录组鉴定和分析,旨在丰富中蜂的miRNA信息,并为深入研究miRNA调控中蜂幼虫肠道发育的分子机理提供依据。【方法】利用sRNA-seq技术对中蜂4、5和6日龄幼虫肠道样品(Ac1、Ac2和Ac3)进行测序,通过数据质控获得有效标签序列(clean tags)。采用Blast工具将clean tags连续比对东方蜜蜂(Apis cerana)基因组和miRBase数据库,以鉴定保守miRNA和新miRNA。采用TPM法对miRNA的表达量进行归一化处理。通过GraphPad Prism7软件统计各组肠道样品中sRNA占比、miRNA长度分布及首位碱基偏向性。利用相关软件预测上述miRNA靶向的mRNA并进行GO和KEGG数据库注释。进一步根据靶向结合关系构建和分析注释到发育和免疫相关通路的基因及其靶向miRNA的调控网络,并利用Cytoscape软件进行可视化。利用茎环反转录PCR(Stem-loop RT-PCR)、分子克隆和Sanger测序验证miRNA的表达和序列的真实性。【结果】共鉴定到中蜂的371个保守miRNA和64个新miRNA;这些miRNA的长度介于18—25 nt且首位碱基主要偏向于U;上述miRNA共靶向14 750条mRNA,涉及离子结合、金属离子结合、细胞膜、细胞膜组件和单一有机体进程等2 270个GO条目,以及内吞作用、细胞凋亡、mTOR信号通路、RNA转运和昆虫激素的生物合成等332条KEGG通路。进一步分析结果显示156个miRNA与注释到Wnt、Hippo、Notch和mTOR等生长发育相关通路的67个靶基因存在调控关系,145个miRNA与注释到Toll、Imd/JNK、Jak-STAT和抗菌效应因子等免疫相关途径的21个靶基因存在调控关系。Stem-loop RT-PCR结果显示miR-8-y、miR-9-z、miR-14-y、miR-281-y、miR-283-x和miR-306-x均能扩增出预期的特异性片段;Sanger测序结果显示上述6个miRNA的序列与深度测序结果一致。【结论】提供了中蜂miRNA的数
【Objective】In this study, transcriptome-wide identification and analysis of mi RNAs 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 mi RNAs’ expression. The ratio of s RNAs, 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 mi RNAs 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, m TOR 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 m TOR signal
作者
冯睿蓉
付中民
杜宇
张文德
范小雪
王海朋
万洁琦
周紫彧
康育欣
陈大福
郭睿
史培颖
FENG RuiRong;FU ZhongMin;DU Yu;ZHANG WenDe;FAN XiaoXue;WANG HaiPeng;WAN JieQi;ZHOU ZiYu;KANG YuXin;CHEN DaFu;GUO Rui;SHI PeiYing(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002;Apitherapy Research Institute,Fujian Agriculture and Forestry University,Fuzhou 350002)
出处
《中国农业科学》
CAS
CSCD
北大核心
2022年第1期208-218,共11页
Scientia Agricultura Sinica
基金
国家现代农业产业技术体系建设专项(CARS-44-KXJ7)
福建农林大学杰出青年科研人才计划(xjq201814)、福建农林大学硕士生导师团队项目(郭睿)
福建省大学生创新创业训练计划(202010389012,202010389016)。
