基于致倦库蚊转录组的溴氰菊酯抗性相关基因分析

doi:10.12140/j.issn.1000-7423.2020.04.010

摘要/Abstract

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。

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

Abstract:

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.

Key words: Culex pipiens quinquefasciatus, Deltamethrin, Resistance gene, RNA-seq, Real-time quantitative PCR

中图分类号:

R384.11

沈瑞鑫, 王意婷, 李春晓, 吴家红, 赵彤言, 陈艳. 基于致倦库蚊转录组的溴氰菊酯抗性相关基因分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(4): 453-463.

SHEN Rui-xin, WANG Yi-ting, LI Chun-xiao, WU Jia-hong, ZHAO Tong-yan, CHEN Yan. 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.

图/表 6

表1

表2

图1

图2

表3

RR株、HN株与SS株比较主要差异表达基因

差异表达基因Differentially-expressed genes	RR株 RR strain				HN株 HN strain				注释Annotate
差异表达基因Differentially-expressed genes	倍数变化Fold change		P值P value		倍数变化Fold change		P值P value		注释Annotate
上调表达 Up-regulatedexpression
CPIJ01956	11.967		8.10 × 10^-31		10.614		7.30 × 10^-16		保守假定蛋白Conserved hypothetical protein, mRNA
XP_001851477.1	11.871		1.64 × 10^-30		8.736		2.33 × 10^-7		微管连接蛋白9 Sorting nexin-9
CP020784	11.766		6.05 × 10^-29		5.225		4.63 × 10^-5		信号转导Signal transduction
NC_040576	11.641		4.47 × 10^-28		4.172		9.12 × 10^-95		ABC转运蛋白//RNA解旋酶 ABC transporter//RNA helicase
CPIJ009463	11.159		5.34 × 10^-27		10.798		3.90 × 10^-25		保守假蛋白 Conservative hypothetical protein
PF02517	10.861		1.36 × 10^-25		10.743		1.67 × 10^-24		金属离子结合//水解酶活性 Metal ion binding//hydro-lyase activity
KT002464	10.512		5.51 × 10^-24		10.343		2.59 × 10^-23		DNA模板对氧化还原状态//转录的负调控的响应 Response to redox state//negative regulation of transcription, DNA-templated
CPIJ009422	10.420		7.43 × 10^-60		3.202		1.27 × 10^-12		假定蛋白Hypothetical protein
CP020782.1	10.334		7.70 × 10^-23		10.129		7.38 × 10^-22		Oryzias latipes strain HNI chromosome 4
CP012523.1	10.302		6.18 × 10^-23		8.069		1.68 × 10^-12		Drosophila busckii chromosome 2L sequence
CPIJ004172	10.293		4.59 × 10^-45		10.22		5.78 × 10^-43		苏糖基-tRNA合成酶 Threonyl-tRNA synthetase
CPIJ009661	10.230		1.65 × 10^-24		5.217		1.20 × 10^-12		保守假定蛋白 Conserved hypothetical protein
KT002466.1	10.220		7.28 × 10^-23		9.614		7.48 × 10^-20		乙酰胆碱酯酶等位基因 Culex quinquefasciatus acetylcholinesterase gene, ace1R2,ace1R2large allele
KXJ79544.1	10.216		6.38 × 10^-23		10.373		7.50 × 10^-23		假设蛋白（RP20_CCG000387 Hypothetical protein (RP20_CCG000387）
CPIJ012473	10.206		7.35 × 10^-23		10.614		7.30 × 10^-16		微管结合蛋白 Microtubule binding protein
CPIJ001064	10.172		2.70 × 10^-22		10.407		3.51 × 10^-6		锌指蛋白596-样 Zinc finger protein 596-like
XP_012565408.1	10.125		8.40 × 10^-9		10.343		2.59 × 10^-23		非活性菱形蛋白1 Inactive rhomboid protein 1
CPIJ008871	10.123		1.19 × 10^-22		8.280		4.61 × 10^-8		假定蛋白Conserved hypothetical protein
CPIJ010145	9.947		8.22 × 10^-22		9.228		1.59 × 10^-18		Culex quinquefasciatus midasin
CPIJ001172	9.942		8.16 × 10^-22		10.107		3.19 × 10^-22		Culex quinquefasciatus l-caldesmon
LR584403.1	9.934		1.22 × 10^-21		9.479		1.27 × 10^-19		Rhinatrema bivittatum genome assembly, chromosome: 17
CP050460.1	9.931		8.56 × 10^-23		9.533		2.27 × 10^-21		Leeuwenhoekiella sp. ZYFB001 chromosome
下调表达 Down-regulated expression
AY958427	16.842		1.94 × 10^-59		15.882		4.08 × 10^-56		胰凝乳蛋白酶 Chymotrypsin
CPIJ000835	15.749		1.60 × 10^-52		10.412		1.32 × 10^-206		胰凝乳蛋白酶2 Chymotrypsin 2
AF468495.1	15.439		2.28 × 10^-50		11.585		2.25 × 10^-112		胰凝乳蛋白酶2 Chymotrypsin 2
CPIJ015368	15.017		3.12 × 10^-48		14.773		1.22 × 10^-46		翻译控制的肿瘤蛋白 Translation-controlled tumor protein
CPIJ001132	14.845		2.85 × 10^-46		12.164		4.34 × 10^-59		延伸因子2 Elongation factor 2
AF420269.1	14.751		2.28 × 10^-46		14.506		8.36 × 10^-45		长型D7clu1唾液蛋白 Long D7clu1 salivary protein
CPIJ001132	14.348		1.89 × 10^-44		14.103		5.87 × 10^-43		延伸因子2 Elongation factor 2
CPIJ010145	14.299		1.25 × 10^-42		14.054		4.16 × 10^-41		延伸因子2 Elongation factor 2
CPIJ003009	14.133		1.96 × 10^-42		13.168		5.81 × 10^-37		DNAⅡ型拓扑异构酶（ATP水解）活性DNA type Ⅱ topoisomerase (ATP hydrolysis) activity
AF468495	14.042		2.58 × 10^-42		13.797		7.89 × 10^-41		胰凝乳蛋白酶样蛋白 Chymotrypsin-like protein
差异表达基因Differentially-expressed genes		RR株 RR strain				HN株 HN strain				注释Annotate
差异表达基因Differentially-expressed genes		倍数变化Fold change		P值P value		倍数变化Fold change		P值P value		注释Annotate
XP_018402067		13.926		1.61 × 10^-41		13.681		4.53 × 10^-40		磷脂分解代谢过程//脂质分解代谢过程Phospholipid catabolic process // Lipid catabolic process
CPIJ013279		13.803		6.02 × 10^-41		13.559		1.73 × 10^-39		保守的假定蛋白 Conservative hypothetical protein）
XM_010779802		13.621		5.01 × 10^-39		2.026		2.77 × 10^-3		无 No
AY958426		13.433		1.40 × 10^-142		4.260		1.81 × 10^-6		胰蛋白酶mRNA Trypsin mRNA
AY388545		13.398		9.79 × 10^-39		4.251		4.60 × 10^-11		推定的17 500唾液肽mRNA Putative 17 500 basic salivary protein mRNA
CPIJ001563		13.222		1.19 × 10^-36		12.974		2.90 × 10^-35		ATP合酶γ链、线粒体ATP Synthase gamma chain, mitochondria
CPIJ000835		13.208		2.48 × 10^-37		10.949		8.92 × 10^-38		胰凝乳蛋白酶2 Chymotrypsin 2
CPIJ013914		12.915		8.75 × 10^-36		6.175		3.62 × 10^-36		重组蛋白 Culex quinquefasciatus dullard protein
XM_029112977		12.828		1.75 × 10^-34		5.358		5.28 × 10^-50		黏蛋白Mucin-7
CPIJ010012		12.796		1.22 × 10^-15		11.830		1.15 × 10^-30		假定蛋白Hypothetical protein

表3

图3

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基因名称Gene name	引物（5′→3′） Primer sequence（5′→3′）	产物长度/bp Product length/bp
18S	F：ATTACGTCCCTGCCCTTTGTAC	74
	R：CACCTTCAAAGACCTCACTAA- ATAATCC
	P：CACCGCCCGTCGCTACTACCGA
RPL8	F：AGTTCAAGCTCCGCAAGCA	62
	R：CACGAACTGGCCGGTGTAC
	P：TTCATCGCCGCCGAGGGC
CPIJ004726	F：TACGGCCCTGCACCAATC	73
	R：CGCCAAAGCAGGAAAATCC
	P：CGTCGCAAAACACCACCAGCATA- CAC
CPIJ019750	F：CGCCTATGCACGCAAACC	68
	R：GCCGAACACCTGGATCTATCTT
	P：CACCTTGAAAAACATCGGACCGGC
P45011A1	F：GACATATCTTTCAGTGCTTCGTAG- GT	79
	R：AGAAGACCCGTGCCGAAGTA
	P：TGACCGCCATGAGCTGCCAACA
OBP45	F：CACATTGCGCGTCAAAACC	66
	R：CTGGTGTGGCAGTTGAGATTTC
	P：CCGCTGCGAGCTGTCACTGAGC
GSTd2	F：GCGCTCAATGTTGGCGTACT	65
	R：CCTCGATCACGACCTTCAA
	P：CCAAATCAACTCCGGCGGCCA
NYDOP7	F：CTCACCATCATCTACTCGTACACC- TT	74
	R：GCCTGTTCACGCATCGATT
	P：CCTGAAGGCGGTCTCGGCCC
P4506B1	F：AACCCGCCGCTTGCTT	76
	R：CGAGCTGGCCGTTGATTT
	P：TGACCAAACTGTGCACCAA- GAACTACAGCC
RPS14/S29e	F：AATCTGCAAAGCAACATGTATAA- AGAG	146
	R：CTCGGCACAGTGTTGAACAACT
	P：TCCCCCCACCGCCCTTTCG
Carbonic anhydrase	F：CACAAGTGGCGACGAGGAT
Carbonic anhydrase	R：TTTTCACTTTCTGGCACTGTTTG	91
	P：CGAATCCATCGGGTCCATTCCG	91
OR7a	F：ACTCGAAGAAGCCGTAGCAGTT	67
	R：ACGATGCTGAACGTGATAATCG
	P：AAACCCAACGGTTGCTGCGCC
OR10a	F：ATCGCTACGACCGGGAAGT	57
	R：TCTGAGCTCGAACCATCATCAT
	P：CGCCAGCTCATCCA
CPIJ006795	F：TGGCACCCCTGTGGATATAA	111
	R：TTGGCCACACCCGGAGTA
	P：TCCCCCCGGTAGAACCTTCCCTCA

蚊虫株Strain	LC₅₀值/μg·ml^-1 LC₅₀ value/μg·ml^-1	95%置信区间 95% confidence interval	χ² (df)	P值 P value	抗性倍数 Resistance ratio
SS	0.000 002 9	0.000 001 92~0.000 006 87	9.265(3)	0.026	1
HN	0.014	0.002~0.032	2.521(3)	0.472	4 828
RR	0.572	0.381~0.782	5.224(4)	0.265	197 241