尖音库蚊复合体蚊虫DNA甲基化水平分析

doi:10.12140/j.issn.1000-7423.2024.04.012

摘要/Abstract

摘要：

目的分析比较尖音库蚊复合体中淡色库蚊、致倦库蚊、骚扰库蚊的DNA甲基化水平以及致倦库蚊吸血前后DNA甲基化水平。方法采集羽化后5 d且未吸血的3个库蚊亚种和吸血3 d的致倦库蚊，提取DNA，超声切割为约250 bp的片段后进行测序，测序数据与致倦库蚊基因组序列（Taxonomy：ID7176）进行比对，获取全基因组胞嘧啶碱基甲基化信息。从基因组、染色体和染色体元件水平分析3个库蚊亚种的甲基化水平（甲基化水平高于3%定义为高甲基化）。比较未吸血的3个库蚊亚种间以及吸血前后致倦库蚊的甲基化水平差异，P < 0.001且甲基化差异绝对值> 5的位点记为差异甲基化位点；Q < 0.05且甲基化差异绝对值> 3的区域记为差异甲基化区域（DMR）。将距离DMR最近的转录起始位点（TSS）所在基因记为DMR相关基因，对其进行基因本体论（GO）富集分析。结果淡色库蚊、骚扰库蚊和致倦库蚊全基因组甲基化水平分别为0.454%~0.672%、0.491%~0.649%和0.499%~0.655%（均低于3%），CHH位点甲基化水平分别为0.631%、0.618%和0.624%，均高于CHG位点的0.567%、0.559%、0.559%（t = 7.14、83.43、6.87，均P < 0.05）和CG/CpG位点的0.508%、0.505%、0.505%（t = 10.59、12.52、13.33，均P < 0.05）；3个库蚊亚种共有的高甲基化位点有56个、高甲基化区域有11个。淡色库蚊、骚扰库蚊和致倦库蚊之间全基因组甲基化水平差异无统计学意义（F = 0.07，P > 0.05），1号染色体（0.568%、0.562%、0.565%）、2号染色体（0.573%、0.564%、0.566%）、3号染色体（0.575%、0.566%、0.569%）的甲基化水平差异均无统计学意义（F = 0.05、0.11、0.13，均P > 0.05），启动子（0.567%、0.552%、0.556%）、外显子（0.562%、0.556%、0.558%）、内含子（0.561%、0.550%、0.555%）和TSS（0.579%、0.506%、0.621%）的甲基化水平差异均无统计学意义（F = 0.37、0.06、0.06、0.16，均P > 0.05）。淡色库蚊和骚扰库蚊间筛选出178个差异甲基化位点、4个DMR，淡色库蚊和致倦库蚊间筛选出209个差异甲基化位点、8个DMR，骚扰库蚊和致倦库蚊间筛选出215个差异甲基化位点、11个DMR。GO富集结果显示，DMR相关基因主要富集于对辐射反应、对光刺激反应和对非生物刺激反应等生物过程。致倦库蚊吸血后全基因组甲基化水平从0.602%升高至0.617%，但差异无统计学意义（t = 1.21，P > 0.05）；吸血前1、2、3号染色体的甲基化水平分别为0.569%、0.569%和0.572%，吸血后上升为0.596%、0.597%和0.600%，但差异均无统计学意义（t = 1.31、1.33、1.30，均P > 0.05）；吸血前启动子、外显子、内含子和TSS的甲基化水平分别为0.557%、0.561%、0.560%、0.552%，吸血后上升为0.585%、0.584%、0.584%、0.594%，但差异均无统计学意义（t = 1.48、1.35、1.20、1.69，均P > 0.05）。致倦库蚊吸血前后基因组间有6个DMR。GO富集结果显示，DMR相关基因在细胞组分上主要富集于内体、囊泡等，在分子功能上主要富集于蛋白结合、小GTP酶结合等。结论淡色库蚊、致倦库蚊、骚扰库蚊全基因组甲基化水平较低，两两亚种间的DMR相关基因主要与对非生物刺激反应生物过程相关。吸血后的致倦库蚊甲基化水平略有升高，吸血前后的DMR相关基因主要和蛋白结合相关。

关键词: 尖音库蚊复合体, DNA甲基化, 全基因组亚硫酸氢盐测序技术

Abstract:

Objective To analyze and compare the DNA methylation levels of three subspecies of Culex pipiens complex, including Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus, and the DNA methylation levels of Cx. p. quinquefasciatus before and after blood-feeding. Methods Mosquitoes of the 3 subspecies were collected at 5 days post-feathering without blood-feeding and Cx. p. quinquefasciatus were collected at 3 days after blood-feeding. DNA was extracted and sonicated into fragments of approximately 250 bp. The fragmented DNA was sequenced, and the data were aligned with the reference genome sequence of Cx. p. quinquefasciatus (Taxonomy ID: 7176). Methylation levels of the 3 subspecies were analyzed at the genomic, chromosomal and elemental levels (the methylation level above 3% was considered hypermethylated). The differences in methylation levels among the 3 subspecies mosquitoes without blood-feeding, and in Cx. p. quinquefasciatus before and after blood-feeding were compared. The sites with P < 0.001 and absolute value of methylation difference > 5 were identified as differentially methylated sites. The regions with Q < 0.05 and the absolute value of methylation difference > 3 were identified as differentially methylated regions (DMR). The genes with the nearest transcription start site (TSS) to DMRs were identified as DMR-associated genes, which were subjected to gene ontology (GO) enrichment analysis. Results The genome-wide methylation levels of Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus were 0.454%-0.672%, 0.491%-0.649% and 0.499%-0.655%, respectively, all were below 3%. The methylation levels of CHH of Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus were 0.631%, 0.618% and 0.624%, respectively, which were higher than CHG (0.567%, 0.559%, 0.559%) (t = 7.14, 83.43, 6.87, all P < 0.05) and CG/CpG (0.508%, 0.505%, 0.505%) (t = 10.59, 12.52, 13.33, all P < 0.05). There were 56 hypermethylated sites and 11 hypermethylated regions present among all 3 subspecies. No significant differences were found among the 3 subspecies (F = 0.07, P > 0.05) at genome-wide methylation levels. No significant differences were found in methylation levels of chromosome 1 (.568%, 0.562%, 0.565%), 2 (0.573%, 0.564%, 0.566%) and 3 (0.575%, 0.566%, 0.569%) among the 3 subspecies at the chromosome level (F = 0.05, 0.11, 0.13, all P < 0.05) nor in the methylation levels of promoters (0.567%, 0.552%, 0.556%), exons (0.562%, 0.556%, 0.558%), introns (0.561%, 0.550%, 0.555%) and TSS (0.579%, 0.506%, 0.621%) among the 3 subspecies at the chromosomal elements level (F = 0.37, 0.06, 0.06, 0.16, all P > 0.05). There were 178 differentially methylated sites and 4 DMRs between Cx. p. pallens and Cx. p. molestus; 209 differentially methylated sites and 8 DMRs between Cx. p. pallens and Cx. p. quinquefasciatus; and 215 differentially methylated sites and 11 DMRs between Cx. p. molestus and Cx. p. quinquefasciatus. GO enrichment analysis revealed that the DMR-associated genes were mainly enriched in the biological processes with response to radiation, light stimuli and abiotic stimuli. The genome-wide methylation levels of Cx. p. quinquefasciatus slightly increased from 0.602% before blood-feeding to 0.617% after blood-feeding, without statistically significant differences (t = 1.21, P > 0.05). The methylation levels of chromosome 1, 2 and 3 in Cx. p. quinquefasciatus were 0.569%, 0.569% and 0.572% before blood-feeding, and were 0.596%, 0.597% and 0.600% after blood-feeding. There were no statistically significant differences before and after blood-feeding (t = 1.31, 1.33, 1.30, all P > 0.05). The methylation levels of the promoters, exons, introns and TSS in Cx. p. quinquefasciatus before blood-feeding were 0.557%, 0.561%, 0.560%, 0.552%, and were 0.585%, 0.584%, 0.584%, 0.594% after blood-feeding, respectively. There were no statistically significant differences before and after blood-feeding (t = 1.48, 1.35, 1.20, 1.69, all P > 0.05). There were 6 DMRs in Cx. p. quinquefasciatus between before and after blood-feeding. GO enrichment analysis showed that the DMR-associated genes were mainly enriched in endosomes and vesicles in cell components, and protein binding or small GTPases binding in molecular functions. Conclusion The genome-wide methylation levels of Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus are relatively low. The DMR-associated genes are mainly related to biological processes that respond to abiotic stimuli. The methylation level of Cx. p. quinquefasciatus slightly increases after blood-feeding, and the DMR-associated genes before and after blood-feeding are mainly involved in protein binding.

Key words: Culex pipiens complex, DNA methylation, Whole-genome bisulfite sequencing

中图分类号:

R384.112

郭思含, 黄新安, 徐寒黎, 李春晓, 刘康康, 邢丹, 赵腾. 尖音库蚊复合体蚊虫DNA甲基化水平分析[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(4): 502-511.

GUO Sihan, HUANG Xin’an, XU Hanli, LI Chunxiao, LIU Kangkang, XING Dan, ZHAO Teng. Analysis on DNA methylation levels in mosquitoes of the Culex pipiens complex[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2024, 42(4): 502-511.

图/表 9

表1

图1

表2

图2

表3

尖音库蚊复合体3个库蚊亚种的DMR

染色体定位 Location on chromosome	起始碱基位点 Start base site	终止碱基位点 Stop base site	甲基化类型 Methylation type	甲基化差异 Methylation difference	到最近TSS的碱基数^d No. bases to the nearest TSS^d	最近TSS所在基因的登录号 Accession number of gene with the nearest TSS
2号染色体 Chromosome 2	5 366 001	5 367 000	CHH	降低^a Decreased^a	6 433	XM_038256247.1
2号染色体 Chromosome 2	122 587 001	122 588 000	CHG	降低^a Decreased^a	-231 643	XM_038252708.1
3号染色体 Chromosome 3	45 435 501	45 436 500	CHH	降低^a Decreased^a	69 773	XM_038262706.1
3号染色体 Chromosome 3	132 786 001	132 787 000	CHH	升高^a Increased^a	24 831	XM_001846523.2
1号染色体 Chromosome 1	64 204 501	64 205 500	CHH	升高^b Increased^b	74 694	XR_005277976.1
1号染色体 Chromosome 1	82 580 001	82 581 000	CHH	升高^b Increased^b	24 241	XR_005276702.1
1号染色体 Chromosome 1	82 581 001	82 582 000	CHH	升高^b Increased^b	25 241	XR_005276702.1
1号染色体 Chromosome 1	109 703 001	109 704 000	CHH	降低^b Decreased^a	9 213	XM_038252169.1
1号染色体 Chromosome 1	112 072 501	112 073 500	CHH	降低^b Decreased^a	-476	XM_038266895.1
2号染色体 Chromosome 2	41 386 001	41 387 000	CHH	升高^b Increased^b	-2 644	XM_038253624.1
3号染色体 Chromosome 3	45 435 501	45 436 500	CHH	降低^b Decreased^a	69 773	XM_038262706.1
3号染色体 Chromosome 3	133 968 501	133 969 500	CHH	降低^b Decreased^a	-30 775	XR_005278093.1
1号染色体 Chromosome 1	82 580 001	82 581 000	CHH	升高^c Increased^c	24 241	XR_005276702.1
1号染色体 Chromosome 1	82 581 001	82 582 000	CHH	升高^c Increased^c	25 241	XR_005276702.1
1号染色体 Chromosome 1	125 679 001	125 680 000	CHH	升高^c Increased^c	-33 639	XM_038249324.1
2号染色体 Chromosome 2	34 828 501	34 829 500	CHH	降低^c Decreased^c	3 306	XM_001844514.2
2号染色体 Chromosome 2	92 528 501	92 529 500	CHH	降低^c Decreased^c	34 656	XM_001846840.2
2号染色体 Chromosome 2	122 587 001	122 588 000	CHG	升高^c Increased^c	-231 643	XM_038252708.1
3号染色体 Chromosome 3	101 581 001	101 582 000	CHH	降低^c Decreased^c	-15 420	XM_038261608.1
3号染色体 Chromosome 3	101 581 501	101 582 500	CHH	降低^c Decreased^c	-14 920	XM_038261608.1
3号染色体 Chromosome 3	132 786 001	132 787 000	CHH	降低^c Decreased^c	24 831	XM_001846523.2
3号染色体 Chromosome 3	180 910 501	180 911 500	CHG	升高^c Increased^c	-12 275	XM_038259164.1
3号染色体 Chromosome 3	180 911 001	180 912 000	CHG	升高^c Increased^c	-12 775	XM_038259164.1

表3

图3

图4

表4

图5

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基因名称 Gene name	GenBank登录号 GenBank accession number	引物名称 Primer name	引物序列（5'→3'） Primer sequence (5′→3′)
母体蛋白Pumilio Maternal protein Pumilio	XM_038262706.1	LOC6050340-F	CCGAAATCAACGCTACCCGA
母体蛋白Pumilio Maternal protein Pumilio		LOC6050340-R	ATGAACCGAGATCCGTGCTG
程序性细胞死亡因子6-相互作用蛋白 Programmed cell death 6-interacting protein	XM_038261608.1	LOC6052216-F	CAACTGCTCCTACACCACCT
		LOC6052216-R	CCCGGCATCGGCATTATTTG
过氧化物酶体多功能酶2 Peroxisomal multifunctional enzyme type 2	XM_038254158.1	LOC6032467-F	ATAGCGGAGAACGAAGCCAA
过氧化物酶体多功能酶2 Peroxisomal multifunctional enzyme type 2		LOC6032467-R	GCAGTCCAGAACCCATTCCT
DDB_G0282067	XM_038256964.1	LOC119767704-F	GGCTGGTAGAGTTCTGGTGTTG
DDB_G0282067		LOC119767704-R	GCTCCTCGTCGTGCTTGTATC
色素分散因子 Pigment dispersing factor	XM_001846523.2	LOC6036530-F	ACAGGCTGACAGTGGATAAAGAAG
色素分散因子 Pigment dispersing factor		LOC6036530-R	CAGTGACGAGGCAGATTGGTT
胆盐活化脂肪酶 Bile salt-activated lipase	XM_038256247.1	LOC6050171-F	CAGCAAACGACTTCGGGAGA
胆盐活化脂肪酶 Bile salt-activated lipase		LOC6050171-R	ACCGTGAATGAACACCAGCA
TsetseEP样蛋白 TsetseEP-like protein	XM_038250440.1	LOC119766065-F	CATGAGCACGAGTCCGAAGA
TsetseEP样蛋白 TsetseEP-like protein		LOC119766065-R	TCTGCCCTAGCCATTCCCA
ZCCHC4	XM_001863391.2	LOC6046195-F	TCCGACAAGAGATGCCTTCCA
ZCCHC4		LOC6046195-R	TCCGTTCTTTATCGCTTTGCTACC
糖基转移酶2 Glucosyltransferase 2	XM_001841656.2	LOC6030813-F	GAGATCGCCAAGCCATGTTCA
糖基转移酶2 Glucosyltransferase 2		LOC6030813-R	GCCACGCTCTTACCATCAGG
磷酸二酯酶 Phosphodiesterase	XM_038249068.1	LOC6034424-F	CGCCAGCGAAAGAACTGATAAA
磷酸二酯酶 Phosphodiesterase		LOC6034424-R	CGAACGTAGGCTTGCCAGAA
过氧化氢酶 Catalase	XM_038266148.1	LOC6038874-F	GAGGATGGCGTGTGGGATTT
过氧化氢酶 Catalase		LOC6038874-R	GTTACGCTTCTGGGTGTGGA
核糖体蛋白L32 Ribosomal protein L32		RPL32-F	AAGCCGAAAGGTATCGACAA
核糖体蛋白L32 Ribosomal protein L32		RPL32-R	CAGTAGACGCGGTTCTGCAT