CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >
Analysis of deltamethrin resistance-related genes based on the transcriptome of Culex pipiens quinquefasciatus
Received date: 2020-01-17
Online published: 2020-09-09
Supported by
National Science and Technology Major Project(2017ZX10303404)
Objective To screen for differentially-expressed genes related to deltamethrin resistance by transcriptome analysis of deltamethrin-susceptible and -resistant strains of Culex pipiens quinquefasciatus.Methods Bioassays were performed on deltamethrin-susceptible (SS) strain, Hainan (HN) strain, and deltamethrin-resistant (RR) strain of Cx. pipiens quinquefasciatus. 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.
Rui-xin SHEN , Yi-ting WANG , Chun-xiao LI , Jia-hong WU , Tong-yan ZHAO , Yan CHEN . Analysis of deltamethrin resistance-related genes based on the transcriptome of Culex pipiens quinquefasciatus[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020 , 38(4) : 453 -463 . DOI: 10.12140/j.issn.1000-7423.2020.04.010
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