靶向调控蚊虫CYP450s基因的miRNA鉴定及分析

doi:10.12140/j.issn.1000-7423.2023.06.004

摘要/Abstract

摘要：

目的筛选并鉴定靶向调控蚊虫细胞色素P450s酶系（CYP450s）基因的miRNA，分析差异表达miRNA及其靶基因的功能。方法采集氯氰菊酯敏感（CS）和抗性（CR）品系的淡色库蚊Ⅲ、Ⅳ龄幼蚊各10只、羽化后3 d未吸血雌成蚊5只，分别提取总RNA并测序，采用DEGseq软件计算CS和CR品系miRNA的表达差异。使用RNAhybrid软件和miRanda软件筛选差异表达miRNA的靶基因，并进行基因本体论（GO）显著性富集分析和京都基因与基因组百科全书（KEGG）通路富集分析。选取5个可能调控CYP450s基因表达的差异表达miRNA（miR-11-5p、miR-317_3、miR-278-3p_1、miR-8-5p、miR-305-3p_5）进行实时荧光定量PCR（qRT-PCR）验证测序数据的准确性。qRT-PCR检测5个差异表达miRNA靶基因（CYP6a8、CYP6BB1v2、CYP6N22、CYP6N26P、CYP9b2）的相对表达水平。结果测序结果显示，CS、CR品系淡色库蚊间差异表达的miRNA共有196个，只在Ⅲ龄幼蚊中差异表达的占16.3%（32/196），只在Ⅳ龄幼蚊中差异表达的占20.4%（40/196），只在雌成蚊中差异表达的占26.0%（51/196），在3个阶段都差异表达的miRNA占12.2%（24/196）。Ⅲ龄幼蚊阶段差异表达的91个miRNA中，有45个上调、46个下调；Ⅳ龄幼蚊阶段差异表达的104个miRNA中，有59个上调、45个下调；雌成蚊阶段差异表达的98个miRNA中，有44个上调、54个下调。GO显著性富集分析和KEGG通路富集分析结果显示，差异表达miRNA的靶基因主要与细胞途径、代谢途径、单细胞生物途径等相关，分子功能主要为结合、催化活性、转运体活性等，主要富集在信号传输、传染性疾病、癌症、寄生虫病等相关通路。qRT-PCR结果显示，5个差异表达miRNA的上调、下调趋势与测序结果基本一致，表达情况与其对应的靶基因（CYP6a8、CYP6BB1v2、CYP6N22、CYP6N26P和CYP9b2）的表达情况相反。结论本研究所筛选的5个差异表达miRNA（miR-11-5p、miR-317_3、miR-278-3p_1、miR-8-5p、miR-305-3p_5）能够通过调节CYP450s基因表达来调控淡色库蚊的抗药性。

关键词: 淡色库蚊, 氯氰菊酯, miRNA, 抗药性, 高通量测序

Abstract:

Objective To screen and identify the microRNAs (miRNAs) targeting cytochrome P450 enzymes (CYP450s) genes in mosquitoes, and analyze the differentially expressed miRNAs and the function of their target genes. Methods Ten larvae of each cypermethrin-susceptible (CS) and cypermethrin-resistant (CR) line Culex pipiens pallens mosquitoes and 5 blood-unfed female adult mosquitoes of each line on 3 d post-emergence were collected for extracting total RNA followed by sequencing, to calculate the differential expression between CS and CR lines using DEGseq software. MiRanda and RNAhybrid databases were used to screen the target genes of differentially expressed miRNAs, and gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis was performed. Five differentially expressed miRNAs (miR-11-5p, miR-317_3, miR-278-3p_1, miR-8-5p and miR-305-3p_5) that may regulate the expression of CYP450s genes were selected for real-time quantitative PCR (qRT-PCR) to validate the sequence output. The relative expression level of differentially expressed miRNAs target genes (CYP6a8, CYP6BB1v2, CYP6N22, CYP6N26P, CYP9b2) were detected by qRT-PCR. Results A total of 196 miRNAs were differentially expressed between CS and CR line Culex pipiens pallens. Among them, 16.3% (32/196) were differentially expressed in age Ⅲ larvae, 20.4% (40/196) in age Ⅳ larvae and 26.0% (51/196) in female adult mosquitoes exclusively; 12.2% (24/196) were differentially expressed in all stages. 45 were up-regulated and 46 were down-regulated of 91 miRNAs differentially expressed at age Ⅲ larvae. 59 were up-regulated and 45 were down-regulated of 104 miRNAs differentially expressed at age Ⅳ larvae. 44 were up-regulated and 54 were down-regulated of 98 miRNAs differentially expressed at female adult mosquitoes. The results of the GO and KEGG enrichment analysis showed that the target genes of differentially expressed miRNAs were mainly related to cellular, metabolic and single-cell biological pathways. The molecular functions of target genes were mainly involve with binding, catalytic activity and transporter activity. The target genes were mainly enriched in the pathways related to signal transduction, infectious diseases, cancers and parasitic diseases. The qRT-PCR results showed that the trends of 5 differentially expressed miRNAs were basically consistent with the sequencing results; The expression profiles of 5 miRNAs were opposite to the target genes (CYP6a8, CYP6BB1v2, CYP6N22, CYP6N26P and CYP9b2). Conclusion The five differentially expressed miRNAs screened in this study (miR-11-5p, miR-317_3, miR-278-3p_1, miR-8-5p and miR-305-3p_5) could regulate the insecticide resistance of Culex pipiens pallens by modulating the expression of CYP450s.

Key words: Culex pipiens pallens, Cypermethrin, miRNA, Insecticide resistance, High-throughput sequencing

中图分类号:

R384.1

武佳慧, 宋晓, 程鹏, 刘宏美, 郭秀霞, 王海防, 公茂庆. 靶向调控蚊虫CYP450s基因的miRNA鉴定及分析[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(6): 683-690.

WU Jiahui, SONG Xiao, CHENG Peng, LIU Hongmei, GUO Xiuxia, WANG Haifang, GONG Maoqing. Identification and analysis of miRNA targeting CYP450s genes in mosquitoes[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(6): 683-690.

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引物名称 Primer name	引物序列（5'→3'） Primer sequence (5'→3')
miR-11-5p-F	GCGAGAACTCCGGCTGTGA
miR-317_3-F	CGTGAACACAGCTGGTGGTAT
miR-278-3p_1-F	CGTCGGTGGGACTTTCGT
miR-8-5p-F	CGCATCTTACCGGGCAGC
miR-305-3p_5-F	CGCGGCACATGTTGGAGTA
miR-UP-R	AGTGCAGGGTCCGAGGTATT
U6-F	GCTTCGGCTGGACATATACTAAAAT
U6-R	GAACGCTTCACGATTTTGCG
CYP6a8-F	CGGATGATACGAAGAGCGACGATG
CYP6a8-R	ACCCAGCCAAATAGAACACGAACG
CYP6BB1v2-F	AGACCTGGAGCTGCGAGAAGTG
CYP6BB1v2-R	TGTTGCCCATCTTTCGGAACTCT
CYP6N22-F	TTGAACGAGATTGCCGCACAGG
CYP6N22-R	ACTTCGAGAACATTCTGCCTTGCC
CYP6N26P-F	CAGCACTTCCACGACCGTTCC
CYP6N26P-R	CTCAGGTTCCGCCACTTGTTCC
CYP9b2-F	GTTGGTGCCTGCTGCGATCTAC
CYP9b2-R	CGAACGGAACTCCACGCTTCTC
β-actin-F	AGGACTCGTACGTCGGTGAC
β-actin-R	TGGTGCCAGATCTTCTCTCCAT