基于PNO基因序列分析隐孢子虫种系发育关系

本文以核基因组的功能蛋白丙酮酸∶NADP^＋氧化还原酶（pyruvate∶NADP^＋ oxidoreductase,PNO）编码基因作为研究对象,对本实验室分离保存的隐孢子虫虫株进行扩增测序,用ClustalX 1.81对扩增序列与GenBank相关参考序列进行比对,用Paup4.0程序中邻接法（Neighbor-joining method,NJ）、最大简约法（Parsimony,MP）和最大似然法（Maximum Likelihood,ML）进行聚类分析,以确定不同隐孢子虫分离株之间的遗传进化关系,并以18S rRNA和HSP70基因构僵的基因树作参照,进而评价PNO这一基因座是否适合作为隐孢子虫基因分型和进化关系分析的基因座。结果表明通过PNO构建的进化树将隐孢子虫分为2大类：Cryptosporidium bailey和C.meleagridis处于一个分枝,C.hominis、C.parvum牛基因型和C.parvum鼠基因型处于另一个分枝上。不同隐孢子虫之间的同源性介于95.0%-100%,能有效区分隐孢子虫不同基因型。因此,PNO基因序列也适合作为隐孢子虫分离株种系发育的遗传标记。

PHYLOGENETIC ANALYSIS OF CRYPTOSPORIDIA ISOLATES BASED ON PNO GENE SEQUENCES

We took nuclear gene that encoded protein pyruvate:NADP oxidoreductase(PNO)as the target,amplified and sequenced the Cryptosporidia strains preserved in our lab.The sequences were aligned with other related sequences deposited in GenBank with Clustal X1.81.Three types of phylogenetic analysis were used on the alignment sequences to assess relationships among the isolates and the distances based neighbor-joining(NJ)analysis,parsimony(MP)analysis and Maximum Likelihood(ML).NJ,MP and ML trees constructed with the program PAUP4.0 were similar.Then phylogentic relationships of different Cryptospoiridum spp.or genotypes were determined.And the phylogenic trees were compared with those drawed from 18S rRNA gene and HSP70 gene to determined whether the PNO gene is suitable for phylogentic analysis of Cryptosporidim or not.The results showed that present phylogentic trees of Cryptosporidium species formed two clades,one clade contained C.baileyi and C.meleagridis,the other calde contained C.hominis,C.parvum cattle genotype and C.parvum mouse genotype.The sequences identity of different Cryptosporidium species ranged from 95.0% to 100%.Similar phylogenic relationships obtained with three different methods showed that the partial PNO gene sequences might be suitable for the phylogentic analysis of Cryptosporidim.