基于核糖体基因分析的中华血吸虫分子种系发生研究

目的　测定中华血吸虫细胞核核糖体基因 r DNA- ITS2和 L SU序列 ,并根据这些序列 ,构建基因树 ,探讨中华血吸虫在裂体属内的系统发生位置。　方法　以 GNT- K法抽提基因组 DNA后 ,用特异引物 ,PCR扩增目的基因。扩增后的 PCR产物经纯化后克隆于载体质粒 p T- adv中再次扩增后 ,提取并纯化质粒 DNA,并以此作模板 ,使用通用测序引物 M13 (F/ R)于 L icor测序仪上进行测序。检索 Gen Bank,查找曼氏血吸虫相关血吸虫两基因序列 ,将有关血吸虫同一基因排序、比较分析后 ,以毛毕吸虫和杜氏小裂体吸虫作为外群 ,使用 PHYL IP和 MEGA以邻接法和最大简约法绘制系统发生树。　结果　克隆并测定了中华血吸虫的 r DNA - ITS2和 L SU ,毛毕吸虫的r DNA- ITS2序列 ,以及根据这些序列构建系统发生树。　结论　中华血吸虫 ITS2和 L SU基因的系统发生树结果一致 ,均提示中华血吸虫归属于亚洲血吸虫种群 ,且中华血吸虫可能是该种群血吸虫类群中较古老的虫种

Study on Molecular Phylogeny of Schistosoma sinensium Based on Nuclear Ribosomal DNA

OBJECTIVE: To determine the phylogenetic relationships between Schistosoma sinensium and other Schistosomatid species using DNA sequence data. Two segments of the nuclear rDNA repeat, the second internal spacer (ITS2) and large subunit (LSU/12S) were selected for sequencing. METHODS: Adult worms stored in 100% methanol were washed 3 times with 0.1 x TE (pH8.0) and the genomic DNA was extracted by the GNT-K method. The target regions were amplified by PCR using specific primers. The PCR products were purified before ligation into the plasmid pT-adv (Clontech). Recombinant plasmids were amplified in E. coli (strain TOP10), extracted and purified using routine methods and then sequenced using M13 primers (F/R) on a Licor long-read auto-sequencer. Sequences of related schistosomes were retrieved from GenBank and aligned with our data in the sequence editor ESEE. Gene trees were constructed in PHYLIP (Version 3.6 alpha July, 2,000) and MEGA (version 2.0 beta build 3) using both Maximum Parsimony and Neighbor-Joining methods. For parsimony analysis, all characters were treated as unordered and with equal weights. At least 3,000 cycles of bootstrapping were carried out. For analysis in MEGA, all gap columns were deleted. Schistosomatium douthitti and Trichobilharzia were used as outgroups. RESULTS: The ITS2 and LSU sequences of Schistosoma sinensium were obtained. The ITS2 sequence of Trichobilharzia sp. was reported here for the first time. CONCLUSION: The phylogenetic trees from these data of nuclear rDNA suggested that S. sinensium belongs to the Asian schistosome group. And this species might be an ancient member in the Asian clade.