蜜蜂球囊菌中长链非编码RNA的调控作用  

Regulatory Function of Long Non-Coding RNAs in Ascosphaera apis

作　　者：周丁丁 范元婵 王杰 蒋海宾 祝智威 范小雪 陈华枝 杜宇 周紫彧 熊翠玲 郑燕珍 付中民 陈大福 郭睿 ZHOU DingDing;FAN YuanChan;WANG Jie;JIANG HaiBin;ZHU ZhiWei;FAN XiaoXue;CHEN HuaZhi;DU Yu;ZHOU ZiYu;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)

机构地区：[1]福建农林大学动物科学学院(蜂学学院),福州350002

出　　处：《中国农业科学》2021年第1期224-238,共15页Scientia Agricultura Sinica

基　　金：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基金(CXZX2017343);福建省大学生创新创业项目(202010389012)。

摘　　要：【目的】长链非编码RNA(long non-coding RNA,lncRNA)是一类二、三级结构高度保守,长度>200 nt且不具蛋白编码能力的RNA,在转录和转录后水平广泛参与调控剂量补偿、细胞分化和生长发育等生命活动。本研究基于前期获得的蜜蜂球囊菌(Ascosphaera apis,简称球囊菌)菌丝和孢子混合样品的高质量lncRNA组学数据进行球囊菌lncRNA的顺式(cis)作用、反义lncRNA(antisense lncRNA)作用和竞争性内源RNA(competing endogenous RNA,ceRNA)作用的分析和探讨,以期揭示lncRNA在球囊菌中的潜在功能。【方法】基于lncRNA基因在染色体上的位置,预测lncRNA上下游100 kb以内的蛋白编码基因;使用Blast软件将上下游基因比对到GO和KEGG数据库,以获得功能和通路注释。利用LncTar软件对反义lncRNA的靶mRNA进行预测,并使用Blast软件将上述靶mRNA比对到KEGG和eggNOG数据库。利用TargetFinder软件预测lncRNA靶向结合的miRNA及miRNA靶向结合的mRNA,根据靶向结合关系建立lncRNA-miRNA和lncRNA-miRNA-mRNA调控网络,进而通过Cytoscape v3.7.1软件进行调控网络的可视化。利用RT-PCR对调控网络中的lncRNA、靶miRNA和靶mRNA进行表达验证。【结果】共预测出371个lncRNA的5852个上下游基因,可注释到细胞进程、代谢进程和应激反应等48个功能条目,以及新陈代谢途径、次生代谢产物的生物合成和抗生素的生物合成等121条通路,表明部分lncRNA可通过顺式作用调节上下游基因的表达,从而参与调控球囊菌的生长发育及物质能量代谢等基础生命活动。球囊菌的7个lncRNA与7个靶mRNA存在序列互补关系,其中5个mRNA在eggNOG数据库中仅注释为假定蛋白,仅gene3444在KEGG数据库注释为核孔复合体蛋白An-Nup120和假定蛋白,表明上述1个反义lncRNA可能参与调控核孔复合体蛋白的生物合成等生物学过程。此外,共预测出227个lncRNA与73个miRNA之间存在靶向结合关系,其中多数lncRNA(79.02%)仅能结�【Objective】Long non-coding RNAs(lncRNAs),a kind of RNAs with highly conserved secondary and tertiary structures and a length of more than 200 nt,have no protein-coding potential.LncRNAs are widely involved in transcriptional and post-transcriptional regulations,such as dosage compensation,cell differentiation and growing development.The study aimed to perform analyses of cis regulation,antisense lncRNA regulation and competing endogenous RNA(ceRNA)regulation as well as related discussion based on previously obtained high-quality lncRNA omics dataset from mixed samples of Ascosphaera apis mycelia and spores,and further reveal the putative regulatory role of lncRNAs in A.apis.【Method】Protein-coding genes located at 100 kb upstream and downstream of A.apis lncRNAs were predicted on basis of the position of lncRNA genes;these upstream and downstream genes were annotated to GO and KEGG databases using Blast to gain function and pathway annotations.Target mRNAs of lncRNAs were identified using LncTar software and then annotated to KEGG and eggNOG databases.Target miRNAs of lncRNAs and target mRNAs of miRNAs were predicted using TargetFinder,followed by visualization of regulatory networks using Cytoscape v3.7.1.The expressions of partial lncRNAs,target miRNAs and target mRNAs within regulatory networks were validated using RT-PCR.【Result】A total of 5852 upstream and downstream genes of A.apis lncRNAs were predicted.These upstream and downstream genes could be annotated to 48 functional terms such as cellular process,metabolic process and stress response,as well as 121 pathways including metabolic pathways,biosynthesis of secondary metabolites and biosynthesis of antibiotics,which indicated that part of A.apis lncRNAs could regulate the expression levels of upstream and downstream genes via cis-acting function,thus participating in regulation of basic cell activities including material and energy metabolisms.Seven lncRNAs were found to have sequence complementary pairing relationship with seven target mRNAs.

关 键 词：蜜蜂球囊菌 长链非编码RNA 顺式作用 反义lncRNA 竞争性内源RNA 

分 类 号：S895.137[农业科学—特种经济动物饲养]