糠秕马拉色菌酵母态和菌丝态蛋白差异表达的研究

The study of differentially expressed proteins in yeast and mycelial phases of Malassiza furfur

目的通过双向电泳及串联质谱技术鉴定糠秕马拉色菌酵母态及菌丝态差异蛋白,在蛋白水平探讨两态转化机制及致病机理。方法分别诱导糠秕马拉色菌标准株酵母态和菌丝态菌体,利用玻璃珠研磨和超声波破碎细胞壁,三氯乙酸/丙酮沉淀获取总蛋白。双向电泳分离蛋白,PDQuest软件比对找出差异蛋白点。电喷雾串联质谱对差异点进行肽段测序,用Mascot和NCBI的Blast软件经蛋白质数据库鉴定蛋白质。结果经双向电泳分离的糠秕马拉色菌酵母态、菌丝态蛋白各有800多个蛋白点、64个蛋白点表达量有3倍以上差异,其中11个为酵母态特有,9个菌丝态特有。在选取的40个差异点中,成功鉴定出22个点,共16个蛋白。经Mascot和Blast软件检索,有明确功能的蛋白中,肌动蛋白、丝切蛋白等9个蛋白在菌丝态上调,谷胱甘肽转移酶、细胞支架信号蛋白等5个蛋白下调。结论鉴定出16个蛋白分别与细胞代谢、运动、氧化应激等功能相关,为了解糠秕马拉色菌表型转换机制和致病机理提供重要信息。

Objectives To identify differential proteins in the yeast and mycelial phases of Malassezia furfur by using the two-di-mensional electrophoresis（2-DE）protocol and tandem spectrum（ MS/MS）,and to understand the pathogenic mechanism of M. furfur and its morphological switching mechanism at protein level as well Methods The type strain of M. furfur was inoculated in the yeast and mycelial phase media respectively Vortexing with glass beads and ultrasonication were used to break up the cell wall into pieces,and trichloroacetate/acetone precipitation was applied to obtain the total proteins These proteins were separated and vis-ualized by 2-DE,analyzed by PDQuest soft to detect the differentially expressed protein spots Electrospray-tandem spectrum analy-sis combined with homology search by Mascot and NCBIˊs Blast was used to identify proteins Results The stable protein profiles from the mycelial and yeast phases were compared,more than 800 spots were detected respectively Of these,64 spots were regula-ted more than 3-fold,11 were identified only in the yeast phases and 9 were identified only in the mycelial phases Selected 40 dif-ferential spots,22 were identified,corresponding to 16 unique proteins In the identified proteins,9 proteins including actin,cofilin etc were up-regulated and 5 proteins including glutathione transferase,cytoskeletal signaling protein etc,were down-regulated in mycelial phase Conclusions Sixteen protein spots implicated in some cellular processes,such as metobalism,motility,oxidase stress etc were positively identified by experiencements and important information would be provided for understanding the mecha-nisms underlying phenotype transformation of M. furfur and its pathogenesis.