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Background: The zoonotic agent,Toxoplasma gondii,is a world-wide distributed parasite,can infect a broad range of hosts including humans.TgMIC16 is responsible for binding to aldolase,associated with rhomboid cleavage and presence of trafficking signals during invasion.In this study,we examined sequence variation in MIC16 gene among T.gondii isolates from different hosts and geographical regions.The entire genome region of the MIC16 gene was amplified and sequenced,and phylogenetic relationship was reconstructed using Bayesian inference(BI),maximum parsimony(MP)based on the MIC16 gene sequences.Results: The results of sequence alignments showed two lengths of the sequence of MIC16 gene among all the examined 12 T.gondii strains: 4391 bp in TgCatBr5,MAS,and 4394 bp in RH,TgPLH,GT1,PRU,QHO,PTG,PYS,GJS,CTG,TgToucan.Their A+T content ranged from 50.30%to 50.59%.A total of 96 variable nucleotide positions(0.1-0.9%)were identified,including 27 variations in 10 exons and 69 variations in 9 introns.Phylogenetic analysis of MIC16 sequences showed that typical genotypes(Type Ⅰ,Ⅱand Ⅲ)were able to be grouped into their respective genotypes,but also atypical strains could be differentiated from typical strains.Moreover,the three major clonal lineages(Type Ⅰ,Ⅱ and Ⅲ)can be differentiated by PCR-RFLP using restriction enzyme Pst I.Conclusions: Phylogenetic analysis and PCR-RFLP of sequence variability of the MIC16 locus among T.gondii isolates from different hosts and geographical regions allowed the differentiation of three major elonal lineages(Type Ⅰ,Ⅱ and Ⅲ)into their respective genotypes,suggesting that MIC16 gene may provide a potential marker for population genetic studies of T.gondii isolates.