蜜蜂球囊菌菌丝和孢子中长链非编码RNA的比较及潜在功能分析

Comparison and Potential Functional Analysis of Long Non-Coding RNAs Between Ascosphaera apis Mycelium and Spore

【背景】蜜蜂球囊菌(Ascosphaera apis,简称球囊菌)是一种专性侵染蜜蜂幼虫的真菌病原,可引起成年蜜蜂数量和蜂群群势的急剧下降。长链非编码RNA(long non-coding RNA,lncRNA)是一类新近发现的非编码RNA,在表观遗传、细胞周期、剂量补偿等众多生命活动中发挥重要生物学功能。【目的】明确球囊菌菌丝和孢子中lncRNA的数量、种类和表达谱差异,并探究共有lncRNA、特有lncRNA和差异表达lncRNA(differentially expressed lncRNA,DElncRNA)在菌丝与孢子中的潜在功能。【方法】利用基于链特异性建库的lncRNA-seq技术对球囊菌的纯化菌丝(AaM)和纯化孢子(AaS)分别进行测序。根据FPKM(Fragment Per Kilobase of per Million mapped reads)法计算lncRNA在AaM和AaS中的表达水平。通过Venn分析筛选AaM与AaS的共有lncRNA和特有lncRNA。按照P≤0.05且|log2 fold change|≥1的标准筛选AaM vs AaS比较组中的DElncRNA。通过Blast工具将共有lncRNA、特有lncRNA和DElncRNA的上下游基因比对GO和KEGG数据库,以进行功能及通路注释。根据靶向结合关系构建共有lncRNA、特有lncRNA和DElncRNA的竞争性内源RNA(competing endogenous RNA,ceRNA)调控网络并利用Cytoscape软件进行可视化。利用RT-qPCR验证测序数据的可靠性。【结果】AaM和AaS分别测得108614646和105675408条原始读段(raw reads),经严格过滤得到107780032和104621402条有效读段(clean reads),Q20分别为98.76%和98.72%,Q30分别达到95.84%和95.78%。共鉴定到850个lncRNA。AaM和AaS的共有lncRNA为701个,二者的特有lncRNA分别为39和110个。上述共有lncRNA通过顺式作用调控3992个上下游基因,它们涉及细胞进程、代谢进程和催化活性等42个功能条目,以及代谢途径、次生代谢物的生物合成和抗生素的生物合成等117条通路;AaM的特有lncRNA和AaS的特有lncRNA通过顺式作用分别调控243和672个上下游基因。AaM vs AaS比较组包含的255个DElncRNA通过顺式作用调控1479个上下游基因,它们涉及代谢进程、细胞进程和催化活性等41个功能条目,以及代谢途径、次生代谢产物的生物合成和抗生素的生物合成等107条通路。从共有lncRNA、孢子特有lncRNA和DElncRNA中分别预测到41、5和13个微小RNA(microRNA,miRNA)的前体序列。调控网络分析结果显示,菌丝lncRNA、孢子lncRNA形成较为复杂的ceRNA调控网络;菌丝lncRNA可靶向结合8个miRNA,进而调控77个mRNA;孢子lncRNA可靶向结合7个miRNA,进而调控87个mRNA;2个DElncRNA(TCONS00008630与TCONS00009302)可靶向结合miR-4968-y,进而调控10个mRNA。RT-qPCR验证结果显示4个DElncRNA的差异表达趋势与测序结果一致,表明测序数据真实可靠。【结论】共有lncRNA、特有lncRNA和DElncRNA可能通过调控上下游基因的表达,作为miRNA的前体,以及充当ceRNA影响菌丝和孢子的物质和能量代谢、自噬、转录、MAPK信号通路、泛素介导的蛋白水解、蛋白酶体以及次生代谢产物的生物合成等生物学过程,从而调节球囊菌的生长、发育、生殖和致病性。

【Background】Ascosphaera apis is a fungal pathogen that exclusively infects honeybee larvae,causing a sharp decrease of the population of adult honeybees and colony population.Long non-coding RNA(lncRNA),a recently discovered non-coding RNA(ncRNA),plays a vital biological role in various activities such as epigenetics,cell cycle and dose compensation.【Objective】This study aimed to clarify the differences of number,type and expression profile of lncRNAs between A.apis mycelium and spore,and investigate the potential role of the common lncRNAs,specific lncRNAs and differentially expressed lncRNAs(DElncRNAs).【Method】The purified mycelia(AaM)and purified spores(AaS)of A.apis were respectively sequenced using strand specific library-based lncRNA-seq technology.The expression levels of lncRNAs in AaM and AaS were calculated using FPKM(Fragment Per Kilobase of per Million mapped reads)method.Common lncRNAs and specific lncRNAs were filtered out following Venn analysis.DElncRNAs within AaM vs Aa S comparison group were screened out following the standard of P≤0.05 and|log2 fold change|≥1.Upstream and downstream genes of common lncRNAs,specific lncRNAs and DElncRNAs were aligned against GO and KEGG databases to obtain function and pathway annotations.The competing endogenous RNA(ce RNA)regulation networks of common lncRNAs,specific lncRNAs and DElncRNAs were constructed following target binding relationships,followed by visualization using Cytoscape software.RT-qPCR was performed to verify the reliability of the sequencing data.【Result】In total,108614646 and 105675408 raw reads were gained from AaM and AaS,and after strict filtering,107780032 and 104621402 clean reads were obtained,respectively,with Q20 of 98.76%and 98.72%,and Q30 of 95.84%and 95.78%.A total of 850 lncRNAs were identified.Seven hundred and one lncRNAs were shared by AaM and AaS,and there were 39 and 110 specific lncRNAs.Via cis function,these shared lncRNAs could regulate 3992 upstream and downstream genes involving in 42 functional terms such as cellular process,metabolism process and catalytic activity;and 117 pathways such as metabolism pathway,biosynthesis of secondary metabolites and biosynthesis of antibiotics.Specific lncRNAs for AaM and AaS could respectively regulate 243 and 672 upstream and downstream genes.In Aa M vs AaS comparison group,255 DElncRNAs were identified and found to regulate 1479 upstream and downstream genes,which were associated with 41 functional terms including cellular process,metabolism process and catalytic activity;and 107 pathways including metabolism pathway,biosynthesis of secondary metabolites and biosynthesis of antibiotics.Forty one,five and 13 miRNA precursors were predicted from common lncRNAs,specific lncRNAs and DElncRNAs.The result of regulatory network analysis showed the formation of ceRNA networks of mycelium lncRNAs and spore lncRNAs;ten lncRNAs in mycelium could bind to eight miRNAs,further targeting 77 m RNAs;while eight lncRNAs in spore could link to seven miRNAs,further targeting 87 mRNAs;two DElncRNAs including TCONS00008630 and TCONS00009302 could simultaneously target miR-4968-y,further regulating ten mRNAs.The result of RT-qPCR suggested the differential expression trend of four DElncRNAs were in accordance of that in sequencing result,indicating the reliability of our sequencing data.【Conclusion】Common lncRNAs,specific lncRNAs and DElncRNAs are likely to affect material and energy metabolisms,autophagy,transcription,MAPK signaling pathway,ubiquitin-mediated proteolysis,proteasome and biosynthesis of secondary metabolites,by regulating the expression of upstream and downstream genes,serving as miRNA precursors or ceRNAs,thus regulating the growth,development,reproduction and pathogenicity of A.apis.