基于高通量测序技术分析番鸭呼肠孤病毒感染雏番鸭肠道转录组学的变化
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摘要
番鸭呼肠孤病毒(Muscovy duck reovirus,MDRV)感染雏番鸭可导致肠道黏膜受损,造成肠黏膜免疫功能低下甚至丧失。为寻找番鸭呼肠孤病毒感染雏番鸭肠道组织免疫相关的差异表达基因,本研究建立番鸭呼肠孤病毒自然发病模型,在感染后3d、6d、21d采集同居感染组和对照组试验雏番鸭的空肠,对其进行高通量测序,对差异表达基因进行GO功能分类和KEGG数据库分析。结果显示,感染后3d、6d和21d分别筛选出2 297、5 860和3 721个差异表达基因;GO分子功能结果显示,免疫相关差异表达基因主要和免疫球蛋白的产生、T细胞活化和单核细胞趋化性等有关;KEGG通路富集结果显示,差异表达基因主要涉及在吞噬体、细胞溶质DNA传感途径、细胞因子-细胞因子受体相互作用、Toll样受体信号通路、Jak-STAT信号通路和RIG-I受体信号通路;荧光定量RT-PCR对差异表达基因进行验证,其结果与转录组学结果基本一致。本研究为进一步阐明MDRV感染导致雏番鸭肠道损伤和黏膜免疫抑制的机制奠定基础。
Muscovy duck reovirus(MDRV)infection of young Muscovy ducks(MDs)can cause damage to the intestinal mucosa,resulting in low or even loss of intestinal mucosal immune function.To find the differentially expressed genes(DEGs)related to MDRV infection in the intestinal tissues of MDs,a model of natural MDRV disease was established and the jejunum of MDs were collected in cohabitation infection group(CIG)and natural control group(NCG)3,6 and 21 days post-infection(dpi). The jejunum was subjected to high-throughput sequencing. DEGs were screened for gene ontology(GO)functional classification and analysis in the Kyoto Encyclopedia of Genes and Genomes(KEGG)database. A total of 2,297,5,860 and 3,721 DEGs were screened at the 3,6 and 21 dpi.The results of GO molecular function showed that the DEGs related to immune function were associated mainly with the production of immunoglobulins(Igs),T-cell activation,and monocyte chemotaxis. The blast of KEGG showed that the DEGs enriched in phagosome,cytosolic DNA sensing,cytokine–cytokine-receptor interactions,Toll-like receptors,Jak-STAT,and RIG-I-like receptors signaling pathways.The DEGs were verified by Fluorescence quantitative RT-PCR,and the results were basically consistent with that of transcriptomics.These results lay the foundation for further elucidation of the mechanism of intestinal injury and mucosal immune suppression in MDRV-infected MDs.
Muscovy duck reovirus(MDRV)infection of young Muscovy ducks(MDs)can cause damage to the intestinal mucosa,resulting in low or even loss of intestinal mucosal immune function.To find the differentially expressed genes(DEGs)related to MDRV infection in the intestinal tissues of MDs,a model of natural MDRV disease was established and the jejunum of MDs were collected in cohabitation infection group(CIG)and natural control group(NCG)3,6 and 21 days post-infection(dpi). The jejunum was subjected to high-throughput sequencing. DEGs were screened for gene ontology(GO)functional classification and analysis in the Kyoto Encyclopedia of Genes and Genomes(KEGG)database. A total of 2,297,5,860 and 3,721 DEGs were screened at the 3,6 and 21 dpi.The results of GO molecular function showed that the DEGs related to immune function were associated mainly with the production of immunoglobulins(Igs),T-cell activation,and monocyte chemotaxis. The blast of KEGG showed that the DEGs enriched in phagosome,cytosolic DNA sensing,cytokine–cytokine-receptor interactions,Toll-like receptors,Jak-STAT,and RIG-I-like receptors signaling pathways.The DEGs were verified by Fluorescence quantitative RT-PCR,and the results were basically consistent with that of transcriptomics.These results lay the foundation for further elucidation of the mechanism of intestinal injury and mucosal immune suppression in MDRV-infected MDs.
引文
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[19]Villarino A V,Kanno Y,O′Shea J J. Mechanisms and consequences of Jak-STAT signaling in the immune sys-tem[J]. Nat Immunol,2017,18(4):374-384.
[20]Pertovaara M,Silvennoinen O,Isom?ki P. Cytokine-in-duced STAT1 activation is increased inpatients with pri-mary Sj?gren′s syndrome[J]. Clin Immunol, 2016,165:60-67.
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[2] Wang K,Wu L Y,Dou C Z,Guan X,Wu H G,Liu H R. Research advance in intestinal mucosal barrier and pathogenesis of Crohn′s Disease[J]. Gastroenterol Res Pract,2016,2016:1-6.
[3] Turner J R. Intestinal mucosal barrier function in health and disease[J]. Nat Rev Immunol,2009,9(11):799-809.
[4]吴宝成,姚金水,陈家祥,卢惠明,陈枝华.呼肠孤病毒B3分离株感染番鸭的病理组织学研究[J].福建农林大学学报(自然版),2001,30(4):514-517.
[5]陈少莺,胡奇林,程晓霞,陈仕龙,江斌,林天龙,林锋强,朱小丽,程由铨.番鸭呼肠孤病毒病雏番鸭实质器官的超微结构[J].中国兽医学报,2006,26(6):662-664.
[6]黄丽娜,姜慧慧,王洁,高婷,张叶飞,吴宝成,吴异健,黄一帆.猴头菇多糖对呼肠孤病毒感染番鸭肠道黏膜免疫及抗氧化能力的影响[J].畜牧兽医学报,2015,46(9):1671-1677.
[7]吴异健,姜慧慧,黄丽娜,朱二鹏,周五朵,王全溪,吴晓平,李健,吴宝成,黄一帆.番鸭呼肠孤病毒感染对雏番鸭肠黏膜组织结构及肠道免疫细胞的影响[J].中国预防兽医学报,2018,40(5):432-437.
[8]张礼.高通量RNA-seq测序数据的基因表达水平分析研究[D].南京航空航天大学,2016.
[9] Todd E V,Black M A,Gemmell N J. The power and promise of RNA-seq in ecology and evolution[J]. Mol Ecol,2016,25(6):1224-1241.
[10]Wilhelm B T, Marguerat S, Watt S, Schubert F,Wood V,Goodhead I,Penkett C J,Rogers J,B?hler J.Dynamic repertoire of a eukaryotic transcriptome sur-veyed at single-nucleotide resolution[J]. Nature,2008,453(7199):1239-1243.
[11]吴宝成,陈家祥,姚金水,陈枝华,陈文列,李国平,曾显成.番鸭呼肠孤病毒的分离与鉴定[J].福建农业大学学报,2001,30(2):227-230.
[12]吴异健.两种中药多糖对呼肠孤病毒感染番鸭的免疫调节作用[D].福建农林大学,2010.
[13]Livak K J,Schmittgen T D. Analysis of relative gene ex-pression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method.[J]. Methods,2001,25(4):402-408.
[14]黄丽娜,姜慧慧,王洁,高婷,张叶飞,吴宝成,吴异健,黄一帆.猴头菇多糖对呼肠孤病毒感染番鸭肠道黏膜免疫及抗氧化能力的影响[J].畜牧兽医学报,2015,46(09):1671-1677.
[15]郑玲红,吴异健,廖加磊,蔡栋灵.张英扬,朱二鹏,周五朵,吴宝成,黄一帆.猴头菇多糖对MDRV感染番鸭血清中细胞因子及激素水平的影响[J].中国兽医科学,2017,47(2):118-125.
[16]祁保民,陈晓燕,吴宝成,姚金水,张红雷.番鸭呼肠孤病毒诱导的细胞凋亡观察[J].畜牧兽医学报,2010,41(4):495-499.
[17]Wang Q X,Wu Y J,Cai Y L,Zhuang Y B,Xu L H,Wu B C,Zhang Y D. Spleen transcriptome profile of muscovy ducklings in response to infection with muscovy duck reovirus[J]. Avian Dis,2015,59(2):282-290.
[18]Wang Q X,Yuan X Q,Chen Y,Zheng Q L,Xu L H,Wu Y J. Endoplasmic reticulum stress mediated MDRV p10.8 protein-induced cell cycle arrest and apoptosis through the PERK/eIF2αpathway[J]. Front Microbiol,2018,21(9):1327-1338.
[19]Villarino A V,Kanno Y,O′Shea J J. Mechanisms and consequences of Jak-STAT signaling in the immune sys-tem[J]. Nat Immunol,2017,18(4):374-384.
[20]Pertovaara M,Silvennoinen O,Isom?ki P. Cytokine-in-duced STAT1 activation is increased inpatients with pri-mary Sj?gren′s syndrome[J]. Clin Immunol, 2016,165:60-67.
[21]赵赫. TLR3与RIG-I相关信号分子在鸭呼肠孤病毒感染雏鸭免疫器官中表达变化研究[D].华中农业大学,2015.