Study On The Sequence Of Mitochondrial DNA Of Chinese Schistosoma Japonicum

[Objective]To study genomic diversity among the three natural isolates of Schistosoma japonicum in Yunnan province.To investigate intraspecific genetic characteristic about Schistosoma japonicum.To establish foundation for elucidate the population hereditary constitution of Schistosoma japonicum in China and do further study about biologic characteristics of Schistosoma japonicum.[Methods]Cercariaeum of Schistosoma japonicum were obtained from masccline Oncomelania hupensis from Dali,Xizhou,and Yongsheng in Yunnan Province and one laboratory maintained isolates in Jiangsu Province.Then every male rabbit is infected with 200～250 cercariae of Schisosoma japonicum via abdominal skin. Rabbits were killed on the 42nd after infection.Schistosome japonicurn adults were collected from rabbits through perfusion method.Genomic DNA was isolated with kit, and the complete gene fragments of the mitochondrial cytochrome C oxidase subunit 1(CO1)and cytochrome b(Cytb)and NADH dehydrogenase subunit 1(ND1)and NADH dehydrogenase subunit 6(ND6)and NADH dehydrogenase subunit 4L (ND4L)were amplified by PCR using specific primers and sequenced.Program DNAStar and Mega3.1 were used for sequence analysis.[Results]The complete gene fragments of the mitochondrial cytochrome C oxidase subunit 1(CO1),cytochrome b,NADH dehydrogenase subunit 1(ND1),NADH dehydrogenase subunit 6(ND6)and NADH dehydrogenase subunit 4L(ND4L)of Schistosoma japonicum amplified from different geographic strains were respectively obtained,the length of complete gene fragments of the mitochondrial cytochrome C oxidase subunit 1(CO1),cytochrome b,NADH dehydrogenase subunit 1(ND1), NADH dehydrogenase subunit 6(ND6)and NADH dehydrogenase subunit 4L (ND4L)of Schistosoma japonicum amplified from different geographic strains were 1527bp,1116bp,891bp,459bp and 264bp,respectively.Comparison of sequences representing the isolates revealed small inter-isolate sequence variations of 10,6,6, 6 and 2 basic group sites,respectively.The identity were 99.5%～100.0%,99.6 %～100%,99.5%～100.0%,99.1%～100%and 99.2%～100%, respectively.There was one missense mutant in the gene fragments of the mitochondrial cytochrome C oxidase subunit 1(CO1)and cytochrome b respectively, and two missense mutant in the gene fragments of the mitochondrial NADH dehydrogenase subunit 1(ND1)and NADH dehydrogenase subunit 6(ND6) respectively,except NADH dehydrogenase subunit 4L(ND4L).The phylogenetic tree demonstrate:Schistosoma japonicum from three natural isolates in Yunnan Province and one laboratory maintained isolates in Jiangsu Province obviously assemble,have obvious branch with Schistosoma mansoni,Schistosoma haematobium,Schistosoma mekongi and Schistosoma malay,but can't be constituted independent branch with the published sequences of the same mitochondrial gene were obtained from GeneBank.[Conclusion]The sequences have small difference and higher autoploidy;There was one missense mutant in the gene fragments of the mitochondrial cytochrome C oxidase subunit 1(CO1)and cytochrome b,and two missense mutant in the gene fragments of the mitochondrial NADH dehydrogenase subunit 1(ND1)and NADH dehydrogenase subunit 6(ND6),and no missense mutant in the gene fragment of the mitochondrial NADH dehydrogenase subunit 4L(ND4L);The samples still belong to Schistosoma japonicum in this study and have intimate genetic relationship,but no obvious branch,so can't be constituted intraspecies categorization.