Xiao Jing-ying, Cai Lian-shun, Mitsuru Nagataki, Shinji Tokuhiro, Jarilla Blanca R, Masaaki Shimada, Blair David, Takeshi Agatsuma
Kochi Medical School, Kochi University, Kochi 783-5805, Japan.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2010 Aug;28(4):252-6.
To determine the nucleotide sequence of the partial mitochondrial (mt) genome and the order of the mitochondrial protein-coding genes for Schistosoma bovis for analysis of possible phylogenetic position of this species in the genus Schistosoma.
The genomic DNA of adult worms were extracted by the GNT-K method. The target regions were amplified by PCR using a degenerated primer and specific primer. The PCR products were purified before ligating into the pGEM1 T-vector system. Recombinant plasmids were amplified in Escherichia coli, extracted and purified using routine methods. The nucleotide sequences were determined with an ABI PRISM 3100-Avant DNA sequencer using a BigDye Terminators v3.1 Cycle Sequencing Kit (Applied Bio-systems, CA, U.S.A.) with two T-vector specific primers (T7 and SP6). Positive colonies were sequenced with two internal specific primers to obtain the full sequence of each fragment on both strands by means of primer walking. Sequences of related schistosomes were retrieved from GenBank and aligned with our data. Gene trees were constructed using neighbor joining methods.
The nucleotide sequence was determined and the gene order of this region in S. bovis was found as follows: NADHdehydrogenase4 (nad4)-trnQ (Gln)-trnK(Lys)-NADH dehydrogenase 3(nad3)-trnD (Asp)-NADH dehydrogenase 1(nad1). The gene order covering such region of S. bovis was similar to that of the African Schistosoma species, but strikingly different from the Asian species. Phylogenetic trees inferred from the alignment including partial nad4, nad3, partial nad1 and partial nad4+nad3+nad1 sequence for other 8 Schistosoma spp., respectively, revealed that S. bovis is placed proximally to S. haematobium in the African sub-group, which is identical with those placed by gene order in the African clade.
The mtDNA analysis based on mitochondrial DNA sequence and the gene order strongly support the hypothesis that S. bovis belongs to the African schistosome clade rather than the Asian Schistosoma species.
确定牛血吸虫部分线粒体（mt）基因组的核苷酸序列以及线粒体蛋白质编码基因的顺序，以分析该物种在血吸虫属中可能的系统发育位置。
采用GNT-K法提取成虫的基因组DNA。使用简并引物和特异性引物通过PCR扩增目标区域。PCR产物在连接到pGEM1 T-载体系统之前进行纯化。重组质粒在大肠杆菌中扩增，采用常规方法提取和纯化。使用ABI PRISM 3100-Avant DNA测序仪，采用BigDye Terminators v3.1循环测序试剂盒（美国加利福尼亚州应用生物系统公司）和两个T-载体特异性引物（T7和SP6）测定核苷酸序列。用两个内部特异性引物对阳性菌落进行测序，通过引物步移法获得每条链上每个片段的完整序列。从GenBank检索相关血吸虫的序列并与我们的数据进行比对。使用邻接法构建基因树。
确定了核苷酸序列，发现牛血吸虫该区域的基因顺序如下：烟酰胺腺嘌呤二核苷酸脱氢酶4（nad4）-转运RNA谷氨酰胺（trnQ）-转运RNA赖氨酸（trnK）-烟酰胺腺嘌呤二核苷酸脱氢酶3（nad3）-转运RNA天冬氨酸（trnD）-烟酰胺腺嘌呤二核苷酸脱氢酶1（nad1）。牛血吸虫该区域的基因顺序与非洲血吸虫物种相似，但与亚洲物种明显不同。分别根据包括部分nad4、nad3、部分nad1以及其他8种血吸虫的部分nad4+nad3+nad1序列的比对结果推断的系统发育树显示，牛血吸虫在非洲亚组中与埃及血吸虫亲缘关系较近，这与根据基因顺序在非洲分支中的分类一致。
基于线粒体DNA序列和基因顺序的线粒体DNA分析有力支持了牛血吸虫属于非洲血吸虫分支而非亚洲血吸虫物种的假说。
