意大利蜜蜂工蜂中肠的环状RNA及其调控网络分析  

Analysis of circular RNAs and their regulatory networks in the midgut of Apis mellifera ligustica workers

作　　者：熊翠玲[1] 陈华枝 陈大福[1] 郑燕珍[1] 付中民[1] 徐国钧[1] 杜宇[1] 王海朋 耿四海 周丁丁 刘思亚 郭睿[1] XIONG Cui-Ling;CHEN Hua-Zhi;CHEN Da-Fu;ZHENG Yan-Zhen;FU Zhong-Min;XU Guo-Jun;DU Yu;WANG Hai-Peng;GENG Si-Hai;ZHOU Ding-Ding;LIU Si-Ya;GUO Rui(College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China)

机构地区：[1]福建农林大学蜂学学院,福州350002

出　　处：《昆虫学报》2018年第12期1363-1375,共13页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31702190);现代农业产业技术体系建设专项资金(CARS-44-KXJ7);福建省科技计划项目(2018J05042);福建省教育厅中青年教师教育科研项目(JAT170158);福建农林大学科技创新专项基金(CXZX2017342;CXZX2017343)

摘　　要：【目的】环状RNA(circRNA)在可变剪接、转录调控和来源基因的表达调控等方面具有重要功能。本研究旨在分析意大利蜜蜂Apis mellifera ligustica工蜂中肠circRNA的数量、种类、结构特征和作用,并通过构建和分析circRNA的调控网络探索circRNA的调控功能。【方法】在实验室条件下人工饲养意大利蜜蜂工蜂,利用circRNA-seq技术对意大利蜜蜂7和10日龄成年工蜂中肠样品进行深度测序。利用find_circ软件从质控后的数据中预测circRNA。通过BLAST比对GO和KEGG数据库,对circRNA的来源基因进行功能和代谢通路注释。利用TargetFinder软件预测circRNA靶向结合的miRNA及miRNA靶向结合的mRNA,通过Cytoscape v.3.2.1软件对circRNA-miRNA和circRNA-miRNA-mRNA调控网络进行构建及可视化。通过设计背靠背引物和线性扩增引物RT-PCR对预测出的circRNA进行验证。【结果】意大利蜜蜂工蜂中肠样品的测序平均得到136 463 071条clean reads,去除rRNA后各样品的anchor reads均在136 779 122条及以上。共预测出10 833个circRNA,长度主要介于15～1 000 nt;上述circRNA的类型丰富,其中已注释的外显子circRNA数量最多,分布在西方蜜蜂1号染色体的circRNA数量最多,其次为8号染色体。CircRNA的来源基因可注释到包括结合、细胞进程和细胞在内的45个GO条目,以及包括内吞作用、内质网蛋白加工及核糖体在内的121条KEGG代谢通路,表明circRNA在意大利蜜蜂工蜂中肠的生长、发育、新陈代谢和细胞生命活动等生物学过程中发挥重要作用。进一步构建circRNA-miRNA和circRNA-miRNA-mRNA调控网络,分析结果显示部分circRNA可能作为竞争性内源RNA吸附结合microRNA,从而调控基因的表达水平。最后,对随机选择的3个circRNA的RT-PCR结果验证了其真实存在。【结论】本研究对意大利蜜蜂工蜂中肠中的circRNA进行预测、分析及鉴定。研究结果提供了中肠circRNA的数量、种类、结构特�【Aim】Circular RNA(circRNA)plays a key role in alternative splicing,transcription regulation and expression regulation of source genes.This study aims to analyze the quantity,variety,structural characteristics and function of circRNAs in the midgut of Apis mellifera ligustica workers,and to explore the regulatory function of circRNAs via constructing and analyzing regulatory networks.【Methods】A.m.ligustica workers were reared under laboratory conditions,and the midgut samples from 7-and 10-day-old workers were subjected to deep sequencing using circRNA-seq technology.CircRNAs were predicted from sequencing data after quality control using find_circ software.Source genes of these circRNAs were annotated to GO and KEGG databases to gain function and pathway annotations via BLAST.The target mRNAs of circRNAs and miRNAs were predicted with TargetFinder software,and the regulatory networks between circRNAs and miRNAs and between circRNAs,miRNAs and mRNAs were constructed and visualized using Cytoscape v.3.2.1 software.The predicted circRNAs were validated by RT-PCR with the divergent and convergent primers designed.【Results】Sequencing of midgut samples from A.m.ligustica workers produced a mean of 136 463 071 clean reads,and for each sample over 136 779 122 anchor reads were obtained after removing rRNA.A total of 10 833 circRNAs were predicted,and their length ranged mainly from 15 to 1 000 nt.The types of these circRNAs were abundant,and the largest one was annotated to be exonic circRNA.The number of circRNAs distributed on chromosome 1 of Apis mellifera was the most and that distributed on chromosome 8 was the second.The source genes of circRNAs could be annotated to 45 GO terms including binding,cellular process,and cell,as well as 121 KEGG metabolic pathways including endocytosis,protein processing in endoplasmic reticulum,and ribosome,suggesting that circRNAs may play key roles in such biological processes as growth,development,metabolism,and cellular activity in the midgut of A.m.ligustica workers.F

关 键 词：意大利蜜蜂 中肠 生物信息学 环状RNA 调控网络 

分 类 号：S891[农业科学—特种经济动物饲养]