转录组测序分析粉尘螨抗辣根素的基因表达与机制  

RNA sequencing analysis of gene expression in Dermatophagoides farinae post‑exposure to horseradish and the underlying mechanisms

作　　者：许昕洁 李镜 吴伊可 杨凤 单文昕 章治鼎 叶长江 孙恩涛 XU Xinjie;LI Jing;WU Yike;YANG Feng;SHAN Wenxin;ZHANG Zhiding;YE Changjiang;SUN Entao(School of Public Health,Wannan Medical College,Wuhu 241002,Anhui,China;School of Laboratory Medicine,Wannan Medical College,Wuhu 241002,Anhui,China;School of Clinical Medicine,Wannan Medical College,Wuhu 241002,Anhui,China)

机构地区：[1]皖南医学院公共卫生学院,安徽芜湖241002 [2]皖南医学院检验学院,安徽芜湖241002 [3]皖南医学院临床学院,安徽芜湖241002

出　　处：《中国寄生虫学与寄生虫病杂志》2025年第1期112-118,123,共8页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(31870352);博士科研启动经费(WYRCQD2022019)。

摘　　要：目的通过转录组测序分析辣根素作用后粉尘螨差异表达基因(DEG)的变化及作用机制。方法将90个熏蒸瓶随机均分为实验组和对照组。每瓶取80只粉尘螨背部朝下置于熏蒸瓶的胶板上,实验组熏蒸瓶中加入0.25 ml/L的辣根素稀释液25µl,对照组加入石蜡。熏蒸24 h后,每组收集2000只存活螨。实验组和对照组各重复6次,3次用于测序,3次用于实时荧光定量PCR(qPCR)验证。提取粉尘螨RNA并逆转录为cDNA,构建文库并进行转录组测序。测序数据进行质控组装后与非冗余蛋白(NR)、基因本体功能注释(GO)、京都基因与基因组百科全书(KEGG)等数据库进行比对。使用edgeR软件进行差异表达分析,对DEG进行GO与KEGG功能注释与富集。选取粉尘螨在辣根素作用后的DEG,进行qPCR验证,使用R软件对qPCR数据进行方差分析。结果转录组测序获得56625条转录本,总长度为72418610 nt。共获得的39510条单基因簇,比对到NR数据库中的数量最多,为25450个,其次分别为直系同源蛋白分组比对(eggNOG)数据库(17643个)、蛋白质(Swiss-prot)数据库(13338个)、GO数据库(13234个)、KEGG数据库(9445个)。辣根素作用粉尘螨后,共检测到2719个DEG,其中上调基因1185个,下调基因1534个。GO功能注释结果显示,DEG主要与细胞过程、结合与催化活性等功能相关,GO富集分析结果显示,上调基因主要为ATP结合盒转运蛋白G亚家族(ABCG)、细胞色素P450(CYP450)、热休克蛋白、乙酰胆碱受体等;下调基因主要为钙调蛋白、RNA聚合酶等。KEGG注释显示,DEG主要参与运输与分解代谢、信号转导、翻译等过程。KEGG富集分析显示,DEG富集于30条通路,主要富集在核糖体、不同环境中微生物代谢以及次生代谢物的生物合成等通路。qPCR结果显示,实验组UDP-葡萄糖醛酸转移酶(UGT)、多重耐药相关蛋白1(MRP1)基因的相对转录水平分别为1.18±0.22、6.22±0.42,较对照组(0.76±0.30、2.52±1.75Objective To identify differentially expressed genes(DEGs)in Dermatophagoides farinae post-treat-ment with horseradish using RNA sequencing,and to unravel the underlying mechanisms.Methods Ninety fumiga-tion bottles were randomly divided into the experimental and control groups.In each bottle,80 D.farinaes were placed back down on the adhesive plates of the fumigation bottles.D.farinaes were exposed to 25µl of 0.25 ml/L horseradish dilutions in the experimental group,and paraffin in the control group for 24-hour fumigation.Then 2000 survival D.farinae were collected from each group.All experiments were repeated 6 times,with triplicates for se-quencing and others for real-time fluorescent quantitative PCR(qPCR)verification.RNA was extracted from D.farinae and reversely transcribed into cDNA,and a library was constructed for RNA sequencing.Following quality control and assembly,the sequencing data were aligned with non-redundant protein sequence(NR),gene ontology(GO),and Kyoto encyclopedia of genes and genomes(KEGG)databases,and the GO and KEGG functional annotations and enrichment analysis of DEGs were performed with the edge R software.In addition,DEGs identified in D.farinaes post-treatment with horseradish were sampled for qPCR assay,the qPCR data were subjected to analysis of variance with the R software.Results RNA sequencing yielded 56625 transcripts with a total length of 72418610 nt,and a total of 39510 UniGene clusters were yielded,with the highest number of clusters aligned to the NR database(25450 clusters),fol-lowed by the evolutionary genealogy of genes:non-supervised orthologous groups(eggNOG)database(17643 clusters),Swiss-prot database(13338 clusters),GO database(13234 clusters),and KEGG database(9445 clusters),respectively.A total of 2719 DEGs were identified in D.farinae post-treatment with horseradish,including 1185 up-regulated genes and 1534 down-regulated genes.GO annotations showed that the DEGs were mainly related to cellular processes,binding and catalytic activities,and GO enrichment anal

关 键 词：粉尘螨 辣根素 转录组 差异表达基因 

分 类 号：R384.429[医药卫生—医学寄生虫学]