多杀性巴氏杆菌脂多糖刺激羊支气管上皮细胞后的转录组测序与分析  

Transcriptome sequencing and analysis of goat bronchial epithelial cells stimulated by Pasteurella multocida Lipopolysaccharide

作　　者：吴艳茹 陈巧玲 刘志勇 李崇瑞 黄惠娴 翟哲 王雪梅[1] 满初日嘎[1] 杜丽[1] 王凤阳[1] WU Yan-ru;CHEN Qiao-ling;LIU Zhi-yong;LI Chong-rui;HUANG Hui-xian;ZHAI Zhe;WANG Xue-mei;MAN Chu-ri-ga;DU Li;WANG Feng-yang(Key Laboratory of Tropical Animal Reproduction&Breeding and Epidemic Disease Research of Hainan Province/College of Animal Science and Technology,Hainan University,Haikou 570228,China)

机构地区：[1]海南大学动物科技学/海南省热带动物繁育与疫病研究重点实验室/海口市动物基因工程重点实验室,海南海口570228

出　　处：《中国预防兽医学报》2022年第7期704-711,717,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家肉羊产业技术体系(财政部和农业农村部CARS-38);海南省院士创新平台科研专项(YSPTZX202013)。

摘　　要：脂多糖(LPS)是多杀性巴氏杆菌(Pm)关键的毒力因子,在其致病机制中发挥重要作用。为了探究不同血清型Pm LPS对宿主细胞的致病机制,本实验将从D型和A型Pm中提取的LPS(D-LPS和A-LPS)分别以1μg/mL刺激羊支气管上皮细胞6 h后裂解各组细胞,采用Illumina HiSeq测序平台进行转录组测序,将测序数据处理后获得转录水平差异基因,利用GO和KEGG数据库对转录差异基因进行GO功能注释与KEGG富集分析,并利用RTqPCR对转录组测序中的转录差异基因进行验证。结果显示:D-LPS组筛选出转录水平上调的基因62个,下调的基因24个,A-LPS组筛选出转录水平上调的基因27个,下调的基因29个,两组相同转录差异基因6个;转录差异基因的GO功能注释结果显示,D-LPS组中共显著富集到243个GO term,包括146个生物学过程(BP)、44个细胞组分(CC)、53个生物学功能(MF),且显著富集的生物学过程有ATP生物合成过程、NADH脱氢酶活性和RAGE受体结合等;A-LPS组中共显著富集到167个GO term,包括109个BP、19个CC、39个MF,且显著富集的生物学过程有IL-11受体结合、GTP酶活性、磷脂酶A2活性和尿嘧啶分解代谢过程等;KEGG富集分析结果显示,D-LPS组中的转录差异基因显著富集到氧化磷酸化、产热以及ECM-受体相互作用途径等通路,A-LPS组中的转录差异基因显著富集到Ras信号通路以及泛酸和辅酶A生物合成等通路。从上述两组中随机选取13个转录差异基因进行RT-qPCR验证,结果与测序结果基本一致。本研究首次分析了两种不同血清型Pm LPS刺激羊支气管上皮细胞后转录组测序数据的变化,结果表明两种Pm LPS均主要影响羊支气管上皮细胞免疫、细胞增殖分化、能量产生及代谢等信号通路,为研究LPS在Pm对宿主致病机制中的作用奠定了基础,同时也为Pm LPS疫苗的研发提供参考依据。Lipopolysaccharide(LPS) is one of the most critical virulence factor of Pasteurella multocida(Pm), and it plays an important role in Pm’s pathogenic mechanism. To explore the pathogenic mechanism of different serotypes of Pm LPS in host cells, the two LPS of type D and type A Pm(D-LPS and A-LPS) were used to stimulate the bronchial epithelial cells of sheep with 1μg/mL for 6 hours respectively, and then the cells in each group were lysed. Transcriptome sequencing was performed using Illumina HiSeq sequencing platform, and transcriptional differential gene were obtained after sequencing data processing.GO and KEGG databases were used for functional annotation and enrichment analysis of transcriptional differential gene, and RT-qPCR was used to verify transcriptional differential gene in transcriptome sequencing. The results showed that 62 upregulated and 24 down-regulated transcriptional differential gene were screened out in D-LPS group, and 27 transcriptional differential genes were up-regulated and 29 were down-regulated in A-LPS group, and 6 transcriptional differential genes were the same between the two groups. GO function annotation results of transcriptional differential genes showed that a total of 243GO terms were significantly enriched in D-LPS group, including 146 biological processes(BP), 44 cell components(CC) and 53biological functions(MF). The biological processes of enrichment include ATP biosynthetic process, NADH dehydrogenase activity and RAGE receptor binding biological processes. A total of 167 GO terms were significantly enriched in A-LPS group,including 109 BP, 19 CC and 39 MF, and the biological processes of significant enrichment included interleukin-11 receptor binding, GTPase activity, phospholipase A2 activity and uracil catabolic process. KEGG enrichment analysis showed that the transcriptional differential gene in D-LPS group were significantly enriched in oxidative phosphorylation, thermogenesis and ECM-receptor interaction pathways, and the differentially expressed genes in A-LP

关 键 词：多杀性巴氏杆菌 脂多糖 Illumina HiSeq测序平台 转录组 

分 类 号：S857.4[农业科学—临床兽医学]