意大利蜜蜂幼虫肠道在球囊菌胁迫后期的差异表达微小RNA及其靶基因分析
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摘要
【目的】蜜蜂球囊菌Ascosphaera apis(简称球囊菌)是一种特异性侵染蜜蜂幼虫肠道的致死性真菌病原。微小RNA(microRNA,mi RNA)是一种在转录后水平对m RNA进行负调控的关键调控因子。本研究旨在对意大利蜜蜂Apis mellifera ligustica(简称意蜂)幼虫肠道在球囊菌胁迫后期的差异表达mi RNAs(DEmiRNAs)及其靶基因进行深入分析,为揭示DEmiRNAs在胁迫应答中的作用提供重要信息。【方法】利用small RNA-seq(s RNA-seq)技术对正常及球囊菌侵染的意蜂6日龄幼虫肠道(分别表示为Am CK和Am T)进行测序,通过相关生物信息学软件对DEmiRNAs进行预测和分析。利用TargetFinder软件预测DEmiRNAs的靶基因,然后利用Blast软件对靶基因进行GO和KEGG数据库的功能注释。通过Cytoscape软件构建DEmiRNAs与其靶m RNAs的调控网络。通过茎环实时荧光定量PCR(stem-loop RT-qPCR)验证测序数据的可靠性。【结果】Am CK和Am T的测序分别得到9 230 496和10 823 667条有效序列标签; Am CK vs Am T比较组中包含15个上调和6个下调mi RNAs,分别结合3 503和3 252个靶基因,它们可分别注释到40和39个GO terms以及104和99条代谢通路;进一步分析发现调控网络中的17个DEmiRNAs靶向结合116个与丝氨酸蛋白酶相关的m RNAs,14个DEmiRNAs靶向结合54个与泛素介导的蛋白水解相关的m RNAs。Stem-loop RT-qPCR验证结果显示随机选择的4个DEmiRNAs (mi R-251-x,mi R-9277-y,mi R-1672-x和mi R-4968-y)的表达量变化趋势与测序结果一致,表明测序数据真实可信。【结论】本研究率先对意蜂幼虫肠道在球囊菌胁迫后期的DEmiRNAs及其靶基因进行预测与分析,提供了mi RNAs的表达谱和差异表达信息。ame-miR-927b,mi R-429-y和mi R-8440-y等DEmiRNAs可能参与对丝氨酸蛋白酶的调控和对泛素介导的蛋白水解的调控,DEmiRNAs与靶基因之间存在复杂的调控网络关系。DEmiRNAs可参与到意蜂幼虫肠道与球囊菌间的互作。
【Aim】Ascosphaera apis is a deadly fungal pathogen that specially infects the larval gut of honeybees. MicroRNA(miR NA) plays a key negative role in regulation of mR NA at the posttranscriptional level. This study aims to comprehensively analyze the differentially expressed miR NAs(DEmiRNAs) and their target genes in the larval gut of Apis mellifera ligustica during the late stage of A.apis stress,so as to provide important information for revealing the roles of DEmiRNAs in the stress response process. 【Methods】Normal and A. apis challenged 6-day-old larval gut of A. m. ligustica(represented as AmC K and AmT,respectively) were sequenced using small RNA-seq(sR NA-seq)technology,followed by prediction and analysis of DEmiRNAs. Target genes of DEmiRNAs were predicted with TargetFinder,and annotations of target genes in GO and KEGG databases were performed using Blast. The regulation networks between DEmiRNAs and the target mR NAs were constructed using Cytoscape. The reliability of the sequencing data was verified by stem-loop RT-qPCR. 【Results】Deep sequencing of AmC K and AmT samples respectively generated 9 230 496 and 10 823 667 clean tags.There were 15 up-regulated and 6 down-regulated miR NAs in AmC K vs AmT comparison group,binding3 503 and 3 252 target genes,which could be annotated to 40 and 39 GO terms as well as 104 and 99 metabolic pathways,respectively. Further investigation indicated that 17 DEmiRNAs within the regulation networks could bind to 116 serine protease-related mR NAs,and 14 DEmiRNAs were capable of binding to 54 ubiquitin-mediated proteolysis associated mR NAs. The result of stem-loop RT-qPCR showed that the variation trends in the expression levels of randomly selected four DEmiRNAs(miR-251-x,miR-9277-y,miR-1672-x and miR-4968-y) were consistent with those in sR NA-seq data,indicating the credibility and reliability of our sequencing data. 【Conclusion】Our study takes the lead in the prediction and investigation of DEmiRNAs and corresponding target genes in the larval gut of A. m. ligustica during the late stage of A. apis stress,and provides expression patterns and differential expression information of miR NAs. The DEmiRNAs including ame-miR-927 b,miR-429-y and miR-8440-y may be involved in the regulation of serine protease and ubiquitin-mediated proteolysis. Complex regulations exist between DEmiRNAs and their target genes. DEmiRNAs are believed to participate in the interactions between the larval gut of A. m. ligustica and A. apis.
【Aim】Ascosphaera apis is a deadly fungal pathogen that specially infects the larval gut of honeybees. MicroRNA(miR NA) plays a key negative role in regulation of mR NA at the posttranscriptional level. This study aims to comprehensively analyze the differentially expressed miR NAs(DEmiRNAs) and their target genes in the larval gut of Apis mellifera ligustica during the late stage of A.apis stress,so as to provide important information for revealing the roles of DEmiRNAs in the stress response process. 【Methods】Normal and A. apis challenged 6-day-old larval gut of A. m. ligustica(represented as AmC K and AmT,respectively) were sequenced using small RNA-seq(sR NA-seq)technology,followed by prediction and analysis of DEmiRNAs. Target genes of DEmiRNAs were predicted with TargetFinder,and annotations of target genes in GO and KEGG databases were performed using Blast. The regulation networks between DEmiRNAs and the target mR NAs were constructed using Cytoscape. The reliability of the sequencing data was verified by stem-loop RT-qPCR. 【Results】Deep sequencing of AmC K and AmT samples respectively generated 9 230 496 and 10 823 667 clean tags.There were 15 up-regulated and 6 down-regulated miR NAs in AmC K vs AmT comparison group,binding3 503 and 3 252 target genes,which could be annotated to 40 and 39 GO terms as well as 104 and 99 metabolic pathways,respectively. Further investigation indicated that 17 DEmiRNAs within the regulation networks could bind to 116 serine protease-related mR NAs,and 14 DEmiRNAs were capable of binding to 54 ubiquitin-mediated proteolysis associated mR NAs. The result of stem-loop RT-qPCR showed that the variation trends in the expression levels of randomly selected four DEmiRNAs(miR-251-x,miR-9277-y,miR-1672-x and miR-4968-y) were consistent with those in sR NA-seq data,indicating the credibility and reliability of our sequencing data. 【Conclusion】Our study takes the lead in the prediction and investigation of DEmiRNAs and corresponding target genes in the larval gut of A. m. ligustica during the late stage of A. apis stress,and provides expression patterns and differential expression information of miR NAs. The DEmiRNAs including ame-miR-927 b,miR-429-y and miR-8440-y may be involved in the regulation of serine protease and ubiquitin-mediated proteolysis. Complex regulations exist between DEmiRNAs and their target genes. DEmiRNAs are believed to participate in the interactions between the larval gut of A. m. ligustica and A. apis.
引文
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Etebari K,Hussain M,Asgari S,2013.Identification of micro RNAs from Plutella xylostella larvae associated with parasitization by Diadegma semiclausum.Insect Biochem.Molec.Biol.,43(4):309-318.
Evans JD,Aronstein K,Chen YP,Hetru C,Imler JL,Jiang H,Kanost M,Thompson GJ,Zou Z,Hultmark D,2006.Immune pathways and defence mechanisms in honey bees Apis mellifera.Insect Mol.Biol.,15(5):645-656.
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Guo R,Wang HP,Chen HZ,Xiong CL,Zheng YZ,Fu ZM,Zhao HX,Chen DF,2018.Identification of Ascosphaera apis micro RNA and investigation of its regulation networks.Acta Microbiol.Sin.,58(6):1077-1089.[郭睿,王海朋,陈华枝,熊翠玲,郑燕珍,付中民,赵红霞,陈大福,2018.蜜蜂球囊菌的micro RNA鉴定及其调控网络分析.微生物学报,58(6):1077-1089]
He L,Hannon GJ,2004.Micro RNAs:small RNAs with a big role in gene regulation.Nat.Rev.Genet.,5(7):522-531.
Li S,Shen L,Sun LJ,Xu J,Jin P,Chen LM,Ma F,2017.Small RNA-Seq analysis reveals micro RNA-regulation of the Imd pathway during Escherichia coli infection in Drosophila.Dev.Comp.Immunol.,70:80-87.
Li ZY,2013.Proliferation and programmed cell death in the fat body in workers of the Italian honeybee(Apis mellifera ligustica)during postembryonic development.Acta Entomol.Sin.,56(11):1252-1257.[李兆英,2013.意大利蜜蜂工蜂脂肪体胚后发育过程中细胞的增殖和凋亡.昆虫学报,56(11):1252-1257]
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