新生隐球菌变种间ITS序列差异的研究

Sequence diversity of ribosomal internal transcribed spacer （ ITS ） region among Cryptococcus neoformans varieties

目的利用真菌核糖体基因内转录间隔区（ITS）序列分析和系统进化学分析来研究新生隐球菌上海变种的地位及变种间ITS序列的差异。方法采用真菌通用引物ITS1、ITS4对12株新生隐球菌不同变种标准株的ITS片段进行PCR扩增和测序，结合基因库等数据库中11株新生隐球菌标准株的ITS序列．用CLUSTALW1．83软件多重比对分析序列的差别，MEGA3．1软件处理数据绘制系统进化树。结果新生隐球菌变种之间ITS序列存在明显差异，存在6种亚型；gattii变种ITS序列存在4种亚型。中国发现的新生隐球菌上海变种S8012与gattii变种在表型及分子生物学研究均存在显著的差异，但仍属于变种内差异。结论我国发现的上海变种S8012归入gattii变种的ITSC型，原gattii变种RV20186归入gatt／i变种的ITSF型。

Objective To evaluate the status of Cryptococcus neoformans vat. shanghaiensis S8012 and assess sequence diversity of ITS region among C neoformans varieties by sequence analysis and phylogenesis analysis on this region. Methods Universal primers （ ITS1 and ITS4 ） were designed and used to amplify the ITS region （ including 5.8S rRNA gene ） of 12 reference strains of C neoformans varieties by PCR, and the products were sequenced. The resultant sequence was blasted against the sequences of 11 reference strains of C neoformans by CLUSTAL W1.83 software with several databases including Genebank. MEGA3.1 software was applied to process data and draw the phylogenetic tree. Results There were significant differences in the sequence of ITS region among C neoformans varieties. As shown by the sequence analysis of ITS region, six subtypes （ A-F ） were identified in the three varieties of C neoformans and 4 subtypes （ C, D, E and F ） in C neoformans var. gattii. Significant differences were observed in the phenotype and molecular biology between C neoformans vat. shanghaiensis S8012 and vat. gattii, but the diversity was within species variation. Conclusions The C neoformans var. shanghaiensis S8012 isolated from Shanghai, China, belongs to subtype C of C neoformans vat. gattii based on ITS sequence analysis, and the original C neoformans vat. gattii R V20186belongs to subtype F of C neoformans var. gattii.