从相关数据库分别下载刚地弓形虫GT1、ME49、VEG虫株,疟原虫以及44个其他真核生物物种的全蛋白质组氨基酸序列,应用HMMER3软件搜索弓形虫不同虫株及其他物种氨基酸序列,鉴定出弓形虫及其他物种磷酸酶家族蛋白。对所有鉴定出的磷酸酶家族蛋白进行聚类分析,提取相对保守的磷酸酶家族蛋白氨基酸序列,分别用邻接法和最大似然法构建系统进化树。应用Pfam在线工具对鉴定出的弓形虫磷酸酶蛋白的结构域进行注释,并进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析,利用基因芯片数据分析并比较蛋白磷酸酶在不同虫株及ME49虫株不同发育时期的转录水平。共鉴定出64个弓形虫磷酸酶蛋白,分属5个磷酸酶亚家族,其中,磷蛋白磷酸酶家族蛋白11个,酪氨酸磷酸酶样蛋白A家族蛋白1个,天冬氨酸依赖的酪氨酸磷酸酶家族蛋白8个,双特异性磷酸酶家族蛋白9个,Mg ²⁺或Mn ²⁺依赖的磷酸酶家族蛋白35个。系统进化树分析结果显示,蛋白磷酸酶在不同弓形虫虫株间高度保守,仅有2个蛋白磷酸酶无直系同源蛋白,为VEG虫株所特有。GO富集分析结果显示,弓形虫蛋白磷酸酶主要具有磷酸酶、水解酶及催化活性的分子功能,参与调节去磷酸化的生物学过程。芯片分析结果显示,蛋白磷酸酶在弓形虫不同虫株速殖子时期的表达谱相似,虫株间表达水平差异最大的为TG_312200,其在ME49虫株中的表达水平约为GT1和VEG虫株的4倍;部分蛋白磷酸酶的转录水平在ME49虫株不同发育阶段差异显著,如TG_318660在未孢子化的卵囊中低表达,TG_304955在速殖子和缓殖子期高表达,这些在不同发育时期差异表达的蛋白磷酸酶可能在虫体发育过程中发挥重要作用。

The proteomic amino acids of Toxoplasma gondii strains GT1, ME49 and VEG, Plasmodium, and 44 other eukaryote species were downloaded from related databases. The amino acid sequences of various T. gondii strains and other species were searched using the HMMER3 software to identify the phosphatases of. T. gondii and other species. Cluster analysis was performed for all of the identified phosphatases to extract the conserved amino acid sequences of phosphatases, and phylogenetic trees were constructed with the neighbor-joining method and maximum likelihood method, respectively. Meanwhile, the catalytic domains of these identified phosphatases were annotated using the Pfam online tool, and gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. Gene array data were used to analyze and compare the transcription levels of protein phosphatases in different strains and at different developmental stages of ME49 strain. Results showed that 64 phosphatases were identified, which were classified into 5 subfamilies: phosphoprotein phosphatase (PPP) family (11 proteins), protein tyrosine phosphatase-like A (PTPLA) family (1 protein), ASP-dependent tyrosine phosphatase (APP) family (8 proteins), dual-specificity phosphatase (DSP) family (9 proteins) and Mg ²⁺ or Mn ²⁺-dependent protein phosphatase (PPM) family (35 proteins). The bioinformatics analysis revealed that the identified phosphatases mainly possessed phosphatase activity, hydrolase activity and catalytic activity, and participated in the regulation of dephosphorylation. The phylogenetic analysis suggested that the phosphatases were conserved among the T. gondii strains. In addition, similar protein phosphatase expression profiles were found at the tachyzoite stage of different T. gondii strains. The most significant difference was found in TG_312200 among different stains. Its expression in ME49 was 4 times as that in the GT1 strain and VEG strains. However, the transcription levels of some phosphatases varied significantly among various developmental stages of the ME49 strain. For example, TG_318660 transcription was at a low level in unsporulated oocysts while TG_304955 transcription was at a high level in both tachyzoites and bradyzoites. These results suggested that these differentially expressed phosphatases may play essential roles in the development of T. gondii.