感染大片形吸虫水牛脊髓全基因组DNA的甲基化及其功能的分析

Methylation and functional analysis of whole-genome DNA in the spinal cord of Bubalus bubalis infected with Fasciola gigantic

为探究感染大片形吸虫后不同时间水牛脊髓全基因组DNA甲基化的类型、占比及其感染后差异甲基化区域(DMR)涉及的功能及信号通路,本研究将大片形吸虫囊蚴经口灌胃感染8~10月龄水牛,分别于感染后3 d(J01)、10 d(J02)、28 d(J03)、42 d(J04)、70 d(J05)和98 d(J06)采集水牛脊髓,利用全基因组重亚硫酸盐测序技术(WGBS)对基因组DNA的甲基化测序,测序数据经过滤筛选后,采用Bismark软件分析各组水牛脊髓基因组DNA甲基化的类型及其占比(某种类型甲基化序列在该组全部可用测序序列中的占比以及该组某种C类型甲基化的数量在全部C类型甲基化中的占比),并采用weight methylation level对各组水牛脊髓基因组DNA 7个功能区域的甲基化进行聚类分析。WGBS测序结果经过滤后显示,平均每组测序长度为100.29 Gp,Q20%(质量值≥20的碱基占总碱基数的百分比)和Q30%分别达到95%和85%以上,6组样品亚硫酸盐(BS)转化率均大于99%,表明WGBS测序结果准确可靠;各组样品DNA甲基化类型和占比的分析及统计结果显示,J01~J06组基因组分别包含CG、CHG、CHH 3种类型的甲基化(mCG、m CH及m CHH),且以m CG类型占比最多(63.1%~71.7%,80.11%~85.73%),m CHH类型(0.9%~1.2%,11.27%~15.18%)及m CHG类型均较少(1.0%~1.2%,3.00%~4.84%);聚类分析结果显示,上述3种类型的甲基化主要分布在水牛脊髓基因组第1内含子和内部内含子。上述结果表明,感染大片形吸虫后水牛脊髓基因组DNA甲基化类型以m CG为主,且第1内含子和内部内含子的甲基化可能影响该两个区域相关基因的正常表达。利用R软件包分析各组水牛脊髓基因组之间是否存在DMR,并采用基于模型的亚硫酸氢盐测序数据分析(MOABS)筛选并统计各组之间的DMR及DMR的数量;采用DAVID软件分析各组DMR在其相应基因组中的分布;采用R语言对各组的DMR进行GO功能注释和KEEG富集分析。DMR的筛选及统计结果显示,各基因组间均存在DMR,且各组间均以CG类型DMR数量的差异最大。其中,J06与J01(7134个)、J06与J02(7174个)、J06与J03(6743个)、J04与J03组(11611个)之间DMR数量的差异最大,且96.42%~99.04%的DMR分布在基因组基因间区,0.96%~3.59%的DMR分布在基因组启动子区。DMR的GO功能分析显示,各组间CG类型DMR的GO功能注释结果基本相似,主要富集在细胞进程、生物调节、代谢过程、结合及催化活性等生物学过程;KEGG分析结果显示,各组间尤其是在感染后期(J06组和J04组)DMR主要富集在癌症及PI3K-Akt等信号通路中。上述结果表明,在大片吸虫慢性感染的过程中,尤其在感染中后期对水牛脊髓基因组基因间区相关基因的表达有影响,且甲基化的DNA可能主要通过以上两个信号通路影响相关基因的表达,进而影响水牛脊髓的各种生物学功能,最终引起水牛中枢神经系统疾病。这在一定程度阐释了寄生在肝脏的片形吸虫影响宿主中枢神经系统的机制。本研究为深入探究大片形吸虫感染对水牛神经系统的影响机制奠定了实验基础。

In order to explore the types and proportion of genome-wide methylation of Bubalus bubalis spinal cord at different times after infection with Fasciola gigantic,and the functions and signaling pathways involved in differential methylation region(DMRs)after infection.In this study,8 to 10 months old Bubalus bubalis were infected with metacercariae of Fasciola gigantic by oral gavage,and the spinal cords of Bubalus bubalis were collected at 3 days post infection(3dpi,J01),10dpi(J02),28dpi(J03),42dpi(J04),70dpi(J05)and 98dpi(J06),respectively.Whole-genome heavy sulfite sequencing technology(WGBS)was used to sequence the spinal cords genomic DNA methylation and the bismark software was used to analyze the types and proportion of genomic DNA methylation in each group(the proportion of a certain type of methylation sequence in all available sequences in this group and the proportion of the number of certain C type methylation in all C type methylation in the group).Weight methylation level was used to cluster the methylation of seven functional regions in the spinal genomic DNA of Bubalus bubalis in each group.The results of WGBS sequencing showed that the average sequencing length of each group was 100.29Gp,Q20%(the percentage of bases with a quality value≥20 in the total number of bases)and Q30%were more than 95%and 85%,respectively,and the conversion rate of sulfite in 6 groups of samples was more than 99%,indicating that the result of WGBS sequencing was accurate and reliable.The DNA methylation types and proportion analysis and statistical results of each group showed that the genomic methylation types were CG type methylation(mCG),CHG type methylation(mCHG)and CHH type methylation(mCHH).mCG type accounted for the most(63.1%-71.7%,80.11%-85.73%),mCHH type(0.9%-1.2%,11.27%-15.18%)and mCHG type were less(1.0%-1.2%,3.00%-4.84%).Cluster analysis of the genome showed that the three types of methylation were mainly distributed in the first intron and the inner intron of the bubalus bulalis genome.These results suggest that mCG is the main type of DNA methylation in bubalus bulalis spinal cord after infection with Fasciola gigantica,and the methylation of first intron and inner intron may affect the normal expression of related genes in these two regions.The existence of DMRs among spinal cord genomes was analyzed by R software package,and the DMR was screened by model-based bisulfite sequencing data analysis(MOABS),and then the number of DMR was counted.The distribution of DMR in the corresponding genomes of each group was analyzed by DAVID online software,and the GO function annotation and KEEG enrichment analysis of DMR in each group were carried out by R language.The screening and statistical results of DMRs showed that there was DMR among all genomes,and the difference in the number of CG type DMR was the biggest.Among them,the difference in the number of CG type DMR among J06 vs J01,J06 vs J02,J06 vs J03 and J04 vs J03 was the largest,which were 7134,7174,6743 and 11611,respectively.The analysis of the distribution of DMR in the corresponding genome showed that 96.42%to 99.04%of the DMR was distributed in the intergenic region and 0.96%to 3.59%of the DMR was distributed in the promoter region of the genome.The GO functional analysis of DMR showed that the results of GO functional annotation of CG type DMR among the groups were basically similar,which were mainly concentrated in biological process such as cellular process,biological regulation,metabolic process,binding and catalytic activity.KEGG analysis showed that DMR was mainly enriched in cancer signaling pathway and PI3K-Akt signaling pathway in the later stage of infection(J06 group and J04 group).The above results suggest that the process of chronic infection of Fasciola gigantica has a certain effect on the expression of genes related to the intergenic region of Bubalus bubalis spinal cord in the later stage of infection and methylated DNA may affect the expression of related genes mainly through the above two signal pathways,and further affect various biological functions of Bubalus bubalis spinal cord,and finally cause buffalo central nervous system diseases.This to some extent explain the mechanism by which Fasciola gigantic parasitic in the liver affect the central nervous system of the host.This study lays the foundation for further exploration of the mechanism of the impact of fluke infection on the nervous system of Bubalus bubalis.