尖音库蚊复合体蚊虫DNA甲基化水平分析

Analysis on DNA methylation levels in mosquitoes of the Culex pipiens complex

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。

Objective To analyze and compare the DNA methylation levels of three subspecies of Culex pipi⁃ens 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).Methyla‑tion 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.quinquefas⁃ciatus 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 differ‑ences were found among the 3 subspecies(F=0.07,P>0.05)at genome‑wide methylation levels.No significant differ‑ences 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.quinquefascia⁃tus.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.quinquefas⁃ciatus slightly increased from 0.602%before blood‑feeding to 0.617%after blood‑feeding,without statistically signifi‑cant 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 methyla‑tion 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 biologi‑cal 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.