摘要
目的通过对致倦库蚊溴氰菊酯敏感株和抗性株进行转录组分析,筛选可能与抗性相关的差异表达基因。方法对致倦库蚊实验室敏感株(SS株)、海南株(HN株)和溴氰菊酯药筛抗性株(RR株) 3个不同抗性水平的株系进行生物测定,分别构建3个株系的cDNA文库,提取RNA,进行转录组测序。基于FPKM值进行差异性分析,对差异基因进行GO和KEGG功能注释与富集分析。筛选出致倦库蚊不同株系中表达量有显著性差异的基因,并采用实时定量PCR验证。结果生物测定结果显示,与致倦库蚊SS株相比,HN株和RR株的抗性倍数分别达到了4 828倍和197 241倍,为高等抗性水平。RNA-seq测序结果显示,致倦库蚊SS株、HN株和RR株转录组共注释基因转录本197 079个。GO富集分析结果显示,在结合、催化活性、细胞过程、代谢过程、单一生物过程、细胞、细胞连接、细胞器中基因富集数量较多。KEGG富集分析结果显示,在富集度最高的前20个条目中,RR株主要富集于剪接体、核糖体等通路,HN株则主要富集于剪切体、RNA转运、hedgehog信号通路、细胞周期、核苷酸切除修复、DNA复制等方向。基于FPKM值的差异性分析结果显示,致倦库蚊RR株和HN株较SS株均上调的有5 357个,均下调的有10 978个。与溴氰菊酯抗性相关基因的靶向性筛选结果显示,与SS株相比,RR株和HN株注释的细胞色素P450基因、谷胱甘肽转移酶基因、气味结合蛋白基因、气味受体基因、视觉基因、核糖体蛋白基因和碳酸酐酶基因转录本中,均上调的分别为100、17、13、4、16、191和1个,均下调的分别为253、48、43、4、15、504和9个。从上调基因中挑选的12个基因(2个细胞色素P450基因、1个谷胱甘肽转移酶基因、1个视觉基因、1个核糖体蛋白基因、1个碳酸酐酶基因、1个核酸内切酶基因、2个气味结合蛋白基因、3个气味受体基因)进行实时定量PCR验证结果显示,在RR株和HN株中,这12个基因较SS株均显著上调,其中RR株较SS株上调1.62~80.26倍,HN株较SS株上调2.01~189.48倍。相关性分析结果显示,12个目标基因的RNA-seq结果与实时定量PCR结果在HN株和RR株中均呈正相关(r=0.802、0.710)。证明这12个目标基因的实时定量PCR结果与转录组测序结果趋势一致。结论基于高通量测序结果和定量PCR验证分析,致倦库蚊中的某些细胞色素P450基因、谷胱甘肽S转移酶基因、视觉基因、嗅觉蛋白基因可能在其对溴氰菊酯的抗性中起一定作用。
Objective To screen for differentially-expressed genes related to deltamethrin resistance by transcriptome analysis of deltamethrin-susceptible and-resistant strains of Culex pipiens q uinq uefas c iatus.Methods Bioassays were performed on deltamethrin-susceptible(SS) strain,Hainan(HN) strain,and deltamethrin-resistant(RR)strain of Cx.pipiens q uinq uefas c iatus.cDNA libraries of each strain were constructed for transcript sequencing.The differentially-expressed genes obtained by differential analysis base on the FPKM value(number of fragments per kilobase of transcript sequence per millions base pairs sequenced) were subjected to GO enrichment analysis and KEGG(kyoto encyclopedia of genes and genomes) functional annotation and enrichment analysis.Genes with significant differences in expression level were screened out and verified by real-time quantitative PCR.Results The resistance levels of the HN and RR strains were 4 828 and 197 241 folds of the SS strain,respectively,both showing high resistance to deltamethrin.A total of 197 079 transcripts were annotated among the transcriptomes of the three Cx.pipiens quinquefasciatus strains.The results of GO enrichment analysis showed enrichment of genes in terms of binding,catalytic activity,cellular process,metabolic process,single organism process,cellular process,metabolic process,cell,cell junction,and organelle.The results of KEGG enrichment analysis showed that among the top 20 items,the RR strain showed enrichment of genes in terms of spliceosome and ribosome pathways,while the HN strain showed significant enrichment of genes in spliceosome,RNA transport,hedgehog signaling pathway,cell cycle,nucleotide excision repair,and DNA replication.Results of differential analysis based on FPKM value showed that there were 5 357 up-regulated and 10 978 down-regulated genes in both the RR and HN strains,compared to the SS strain.The targeting screening for genes related the genes with differential expression in RR and HN strains including cytochrome P450(P450),glutathione transferase(GST),odorant binding protein(OBP),odorant receptor(OR),opsin gene,ribosomal protein gene,and carbonic anhydrase,in comparison with the SS strain.The annotated transcriptomes of these differential genes in RR and HN strains were found having 100,17,13,4,16,191 and 1 up-regulated transcripts,and 253,48,43,4,15,504 and 9 down-regulated transcripts,respectively,compared to the SS strain.To verify,twelve up-regulated genes were selected for real-time quantitative PCR,including 2 P450 genes,1 GST gene,1 opsin gene,1 ribosomal protein gene,1 carbonic anhydrase(CA) gene,1 nucleic acid endonuclease gene,2 OBP genes and 3 OR genes.The results showed that in RR and HN strain,the 12 genes were all significantly up-regulated compared to the SS strain,among them the expressions of those in the RR strain were up-regulated by 1.62-80.26 folds,and those in the HN strain up-regulated by 2.01-189.48 folds.The correlation analysis on the 12 targeting genes showed a positive correlation between the results of RNA-seq and the results of real-time quantitative PCR for NH and RR strain,with a correlation coefficient r of 0.802 and 0.710,respectively.It demonstrates that for the 12 genes,the results of real-time quantitative PCR were consistent with the transcriptome sequencing data.Conclusion Based on the high-throughput sequencing results and quantitative PCR verification analysis,some genes including cytochrome P450 genes,glutathione S-transferase genes,visual genes,and olfactory protein genes in Cx.pipiens quinquefasciatus may play certain roles in the deltamethrin resistance.
作者
沈瑞鑫
王意婷
李春晓
吴家红
赵彤言
陈艳
SHEN Rui-xin;WANG Yi-ting;LI Chun-xiao;WU Jia-hong;ZHAO Tong-yan;CHEN Yan(Department of Parasitology,Besic Medical College,Guizhou Medical University,Guiyang 550000,China;Beijing Institute of Microbiology and Epidemiology,State Key Laboratory of Pathogen and Biosecurity,Beijing Key Laboratory of Vector Borne and Natural Focus Infectious Disease,Beijing 100071,China)
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2020年第4期453-463,共11页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家科技重大专项(No.2017ZX10303404)。
关键词
致倦库蚊
溴氰菊酯
抗性基因
转录组测序
实时定量PCR
Culex pipiens quinquefasciatus
Deltamethrin
Resistance gene
RNA-seq
Real-time quantitative PCR
