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

作　　者：钟舒红 盛兆安 李军 彭昊 吴翠兰 马春霞 彭红艳 黄维义[2] 施维[2] 潘艳 ZHONG Shu-hong;SHENG Zhao-an;LI Jun;PENG Hao;WU Cui-lan;MA Chun-xia;PENG Hong-yan;HUANG Wei-yi;SHI Wei;PAN Yan(Guangxi Veterinary Research Institute,Guangxi Key Laboratory of Veterinary Biotechnology,Key Laboratory of China(Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs of China,Nanning 530001,China;College of Animal Science and Technology,Guangxi University,Nanning 530004,China)

机构地区：[1]广西壮族自治区兽医研究所,广西兽医生物技术重点实验室,农业农村部中国(广西)-东盟跨境动物疫病防控重点实验室,广西南宁530001 [2]广西大学动物科学技术学院,广西南宁530004

出　　处：《中国预防兽医学报》2023年第3期253-263,共11页Chinese Journal of Preventive Veterinary Medicine

基　　金：广西重点研发计划(桂科AB16380106);广西基本科研业务费专项(桂科专项17-3)。

摘　　要：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

关 键 词：水牛 大片形吸虫 脊髓 DNA甲基化 全基因组重亚硫酸盐测序技术 差异甲基化区域 

分 类 号：S852.73[农业科学—基础兽医学]