“夜逸蚴”日本血吸虫线粒体基因组特征与系统进化的关系

Characterization of the mitochondrial genome and phylogenetic implication of Schistosoma japonicum featured with“nocturnal cercarial emergence”

目的测定具有“夜逸蚴”特征的日本血吸虫线粒体全基因组序列,并探讨其与大陆其他日本血吸虫地域株的系统进化关系,为后续进行种群遗传结构与遗传多样性研究提供新的数据。方法2020年于安徽省石台县现场采集钉螺,实验室内分离感染性钉螺,逸出的尾蚴以腹部贴片法感染ICR小鼠,灌注法获取肝门静脉和肠系膜静脉处虫体,随机抽样进行测序。使用血液/组织基因组DNA提取试剂盒提取血吸虫DNA,采用Illumina NovaSeq 6000测序平台进行双末端测序,采用GetOrganelle软件组装线粒体基因组,使用MEGA 11软件分析线粒体基因组结构特征与碱基组成。选择已发表的中国地区17个日本血吸虫线粒体基因组序列,采用最大似然法(ML)和邻接法(NJ)构建系统进化树。结果安徽省石台县分离的“夜逸蚴”日本血吸虫线粒体基因组全长14085 bp(GenBank登录号:ON637109),编码36个基因,由12个蛋白编码基因(PCG),22个tRNA基因和2个rRNA基因组成。线粒体基因组A+T含量为71.35%,存在基因重叠区2处、基因间隔区29处;在22个tRNA基因中,除trnaC和trnaS1基因缺失二氢尿嘧啶(DHU)臂外,其余均能形成典型三叶草二级结构。蛋白编码基因中,cytb、nad4、nad2和nad6基因的终止密码子为TAA,不同于以TAG为终止密码子的中国其他地域株。基于NJ法和ML法构建的系统进化树具有相似的拓扑结构,石台县“夜逸蚴”日本血吸虫分离株单独成一支;中国台湾血吸虫株与中国大陆其他地域株分离,形成一支。结论安徽省石台地区的日本血吸虫分离株其终止密码子与中国其他地域株存在差异,线粒体基因组的系统进化地位分析,与其他地域株亲缘关系较远,遗传差异较大。

Objective This study aimed to determine the complete mitochondrial genome sequence of Schistosoma japonicum featured with nocturnal cercarial emergence pattern and to explore its phylogenetic relationship with other S.japonicum geographical isolates from China's Mainland,providing new reference data for further study on population genetic structure and genetic diversity.Methods Field Oncomelania hupensis snails were collected from Shitai County of Anhui in 2020,and the infected snails were separated in the laboratory.ICR mice were infected with cercariae shed from the infected snails using abdominal patch method.The worms were obtained by perfusion from hepatic portal and mesenteric vein and randomly sampled for sequencing.The worm genomic DNA was extracted with blood/tissue DNA extraction Kit,and sequenced at paied-end using Illumina NovaSeq 6000 sequencing platform.The mitochondrial genome was assembled using GetOrganelle tool,and its structural features and base composition were analyzed using MEGA 11 software.The phylogenetic tree was constructed using the neighbour-joining(NJ)and maximum likelihood(ML)method based on 17 published mitochondrial genome sequences of S.japonicum from other parts of China.Results The complete circular mitochondrial genome of S.japonicum with nocturnal cercarial emergence pattern isolated from Shitai County of Anhui was 14085 bp in length(Genbank accession no.ON637109),encoding 36 genes,consisting of 12 protein-coding genes(PCGs),two ribosomal RNA(rRNA)genes,22 transfer RNA(tRNA)genes,with the AT content of 71.35%.There were two gene-overlapping regions and 29 intergenic regions in the mitochondrial genome.Among the 22 tRNA genes,except for trnaC and trnaS1 genes,which missed the dihydrouridine(DHU)arm,the rest were able to form a typical cloverleaf secondary structure.Among the PCGs,the stop codon of cytb,nad4,nad2 and nad6 was TAA,which differed from other geographical strains in China with TAG as the stop codon.Phylogenetic trees constructed based on NJ and ML had similar topologies.The isolated strain of S.japonicum featured with“nocturnal cercarial emergence”in Shitai County,forms a single strain.S.japonicum strains from Taiwan,China and other regions of Chinese Mainland are separated to form one branch.Conclusion The stop codons of S.japonicum isolate from Shitai area of Anhui Province differed from those of other geographical isolates in China.Additionally,based on the phylogenetic analysis,the isolate featured with nocturnal cercarial emergence exhibited a more distant relationship with other regional isolates,showing greater.