Analysis of deltamethrin resistance-related genes based on the transcriptome of Culex pipiens quinquefasciatus

doi:10.12140/j.issn.1000-7423.2020.04.010

Abstract

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

CLC Number:

R384.11

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.

Figures/Tables 6

Table 1

Table 2

Fig. 1

Fig. 2

Table 3

The main differentially-expressed genes of the RR and HN strains compared to the SS strain

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

Table 3

Fig. 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