刘涛华,王颜颜,吕倩,牟丽丽,陈燕,康颖倩
LIU Taohua,WANG Yanyan,LU Qian,MOU Lili,CHEN Yan,KANG Yingqian

• 1:贵州医科大学
• 2:贵州医科大学微生物学教研室

摘要(Abstract)：

目的:比较单一D1/D2区域测定、多位点测序分型技术(MLST)和基质辅助激光解吸-飞行时间质谱技术(MALDI-TOF-MS)在新生隐球菌(Cryptococcus podzolicus)及与之邻近的其他种属的分类研究的稳定性和可靠性。方法:挑选以Cryptococcus podzolicus为主及与之相邻的其他种和属的菌株共20株及1株outgroup模式菌株,运用酵母菌基因组提取试剂盒提取其DNA,PCR扩增5个管家基因(ITS,D1/D2,rpb1,rpb2,tef1)并测序,以Bayesian法(Mr Bayes3.2.1)、最大似然法、邻接法(MEGA 6.0软件)分别构建5个管家基因及D1/D2基因序列的系统发育树;应用MALDI-TOF-MS采集蛋白指纹图谱,在Bruker Daltonics数据库(BDAL)和CBS真菌保藏中心数据库信息的基础上,对20株菌进行聚类分型,构建聚类分型树状图。结果:3种方法所得树状图在种间的层面上,均出现了a、b、c 3个分支;在种内的层面上,特别是a这一分支,均为Cryptococcus podzolicus,单一D1/D2区域所构建的系统发育树分化程度不大,各菌株间无明显的分类多样性,而MLST及MALDI-TOF MS所得到的树状图在种内均出现了明显的分类多样性及亲缘关系的差异性。结论:MLST及MALDI-TOF MS均可作为隐球菌属中菌株分类鉴别中有效的分子生物学方法,能可靠地揭示隐球菌属中各菌种的种间及种内的遗传进化关系,MALDI-TOF-MS比MLST则更为快捷准确。
Objective: To compare and analyze the stability and reliability of multilocus sequence typing( MLST) and matrix-assisted laser desorption / ionization time of flight mass spectrometry( MALDI-TOF-MS) methods in taxonomic study of Cryptococcus,especially for Cryptococcus podzolicus.Methods: Twenty strains including Cryptococcus podzolicus and its adjacent strains were selected for the evaluation in this study; DNA was extracted using yeast genome extraction kit and 5 genes( ITS,D1 / D2,rpb1,rpb2,tef1) were amplified and sequenced. The phylogeny tree was constructed by Bayesian,maximum parsimony and neighbor-joining analyses. The protein fingerprint acquisition parameters were collected by MALDI-TOF-MS method. Clustering classification was conducted for the twenty strains and the phylogenetic tree was constructed based on BDAL and CBS fungi preservation center database. Results: There existed three clusters( a,b and c) in inter species level that can be observed on the phylogenetic trees made by three different molecular methods; In intraspecific level,all Cryptococcus podzolicus strains were classified into Cluster a,in which there was no apparent taxonomic diversity for the strains in the phylogenetic tree constructed by D1 / D2,while the evolution distances among Cryptococcus podzolicus strains were easily identified by both MLST and MALDI-TOF-MS methods.Conclusion: MALDI-TOF-MS methods can be more effective for identification and classification of Cryptococcus species. Moreover,MALDI-TOF-MS is more efficient and accurate than MLST.

关键词(KeyWords)： 多位点测序分型;基质辅助激光解吸飞行时间质谱;新隐球菌;分类法;发育生物学;进化
multilocus sequence typing;matrix-assisted laser desorption /ionization time of flight mass spectrometry;Cryptococcus podzolicus;classification;developmental biology;evolution

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31060006;31260029);; 贵州省社会发展科技攻关项目[黔科合SY字(2011)3017号];; 贵阳市科技局社会发展与民生计划[筑科合同(2011103)16号];; 贵州省国际科技合作计划项目[黔科合外G字(2014)7006]

作者(Author): 刘涛华,王颜颜,吕倩,牟丽丽,陈燕,康颖倩
LIU Taohua,WANG Yanyan,LU Qian,MOU Lili,CHEN Yan,KANG Yingqian

DOI: 10.19367/j.cnki.1000-2707.2016.02.005

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