牛裂体吸虫线粒体DNA序列和基因排序分析

中国寄生虫学与寄生虫病杂志 ›› 2010, Vol. 28 ›› Issue (4): 3-256.

牛裂体吸虫线粒体DNA序列和基因排序分析

肖景莹1，2，蔡连顺2，長瀧充1，徳弘慎治1，Jarilla Blanca R.1，嶋田雅暁3，David Blair4，吾妻健1 *

1 日本高知大学医学部，高知 783-8505；2 佳木斯大学基础医学院，佳木斯 154007；3 日本长崎大学热带医学研究所，长崎 852-8523；4 澳大利亚詹姆斯库克大学热带生物学院，汤斯维尔 QLD 4811

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2010-08-30 发布日期:2010-08-30

Study on Molecular Phylogeny of Schistosoma bovis Based on Mitochondrial DNA Sequence and Gene Order

XIAO Jing-ying1，2，CAI Lian-shun2，Nagataki Mitsuru1，Tokuhiro Shinji1，Jarilla Blanca R.1，Shimada Masaaki3，David Blair4，Agatsuma Takeshi1 *

1 Kochi Medical School，Kochi University，Kochi 783-5805，Japan；2 Basic Medical College，Jiamusi University，Jiamusi 154007，China；3 Institute of Tropical Medicine，Nagasaki University，Nagasaki 852-8523，Japan; 4 School of Tropical Biology，James Cook University，Townsville QLD 4811，Australia

Received:1900-01-01 Revised:1900-01-01 Online:2010-08-30 Published:2010-08-30

摘要/Abstract

摘要： 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?鄄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pGEM1 T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 adenine dinucleotide dehydrogenase subunit 4-1gene， nad4-nad1），其长度为2 214 bp。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。

关键词: 牛裂体吸虫, nad4, nad3, nad1, 基因排序, 系统发生关系

Abstract: Objective 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. Methods　 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， USA） 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. 　 Results　 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., respectivly， 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. Conclusion 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.

Key words: Schistosoma bovis, NADH dehydrogenase 4, NADH dehydrogenase 3, NADH dehydrogenase 1, Gene order, Phylogenetic relationship

肖景莹;蔡连顺;長瀧充;徳弘慎治;JarillaBlancaR.;嶋田雅暁;DavidBlair;吾妻健. 牛裂体吸虫线粒体DNA序列和基因排序分析[J]. 中国寄生虫学与寄生虫病杂志, 2010, 28(4): 3-256.

XIAOJing-ying;CAILian-shun;NagatakiMitsuru;TokuhiroShinji;JarillaBlancaR.;ShimadaMasaaki;DavidBlair;AgatsumaTakeshi*. Study on Molecular Phylogeny of Schistosoma bovis Based on Mitochondrial DNA Sequence and Gene Order[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2010, 28(4): 3-256.