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目的为探讨牛裂体吸虫(Schistosoma bovis)在裂体属内的系统发生位置,测定牛裂体吸虫线粒体基因部分序列,并分析该编码区域的基因序列和基因排序。方法以GNT-K法抽提牛裂体吸虫成虫基因组DNA,用兼并和特异引物扩增目的基因。扩增产物经纯化后克隆于pGEM1T质粒载体,并转化大肠埃希菌。抽提和纯化阳性质粒DNA,并测序。以纯化后的阳性质粒DNA为模板,根据已获得的序列设计内部特异引物,采用引物步移法获得全长目的片段。在GenBank中查找曼氏血吸虫等相关血吸虫线粒体基因序列,作基因排序及比较分析后,以邻接法绘制系统发生树。结果测定了牛裂体吸虫线粒体烟酰胺腺嘌呤二核苷酸脱氢酶亚基Ⅳ～Ⅰ基因序列(nicotinamide adeninedinucleotide dehydrogenase subunit4-1gene,nad4-nad1),其长度为2214bp。分析该编码区基因排序为nad4-trnQ(Gln)-trnK(Lys)-nad3-trnD(Asp)-nad1。牛裂体吸虫在该区域的线粒体基因排序与非洲支系血吸虫相似,与亚洲支系血吸虫有很大的不同;根据牛裂体吸虫与其他8种吸虫部分nad4,nad3,部分nad1和部分nad4+nad3+nad1基因序列比对结果,分别构建系统发生树。结果表明,牛裂体吸虫与埃及血吸虫位于同一簇,归属于非洲血吸虫支系,这与由基因排序推测的牛裂体吸虫的系统发生位置结果相一致。结论牛裂体吸虫属于非洲支系而非亚洲支系血吸虫。
OBJECTIVE: To investigate the phylogenetic position of Schistosoma bovis in the genus Rhesus, and to identify the mitochondrial DNA sequence of the genus Schizonts sinensis, and to analyze the gene sequence and gene sequence of this gene. Methods The GNT-K method was used to extract genomic DNA of adult C. sinensis adults, and the target genes were amplified by merging and specific primers. The amplified product was cloned into pGEM1T plasmid vector and transformed into Escherichia coli. Positive plasmid DNA was extracted and purified and sequenced. Using the purified positive plasmid DNA as a template, internal specific primers were designed according to the sequences obtained, and the full-length target fragment was obtained by primer walking. The gene sequence of mitochondrial genes of Schistosoma mansoni related to Schistosoma mansoni was searched in GenBank for gene sequencing and comparative analysis, and the phylogenetic tree was drawn by the neighbor-joining method. Results The mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 4-1 gene (nad4-nad1) was sequenced and its length was 2214 bp. The coding region was analyzed for the order of nad4-trnQ (Gln) -trnK (Lys) -nad3-trnD (Asp) -nad1. The sequence of mitochondrial genes in this region is similar to that of the African schistosomiasis and is very different from that of the Asian schistosomiasis. According to the sequences of nad4, nad3, some nad1 and some nad4 + nad3 + nad1 gene sequence alignment results were constructed phylogenetic tree. The results showed that T. gondii was located in the same cluster with S. schistosome as belonging to African schistosomiasis, which was consistent with the phylogenetic position of S. occidentalis estimated by gene sequencing. Conclusion S. oxalis belonged to the African clade but not to the Asian clade schistosome.