双向电泳和质谱技术分析樟芝无性孢子萌发相关蛋白

Analysis of germination-related proteins in Antrodia camphorata athroconidia by two-dimensional electrophoresis and mass spectrum

摘要:

[目的]采用双向电泳（2DE）、质谱技术和实时荧光定量PCR （RT-qPCR）技术分析樟芝无性孢子萌发相关蛋白。[方法]分别提取培养0 h和24 h的樟芝孢子总蛋白并进行双向电泳分离，再用PDQuest软件进行差异蛋白分析，并用MALDI-TOF-MS技术对差异蛋白进行鉴定；其次将鉴定成功的蛋白与孢子萌发相关蛋白的本地数据库进行比对，获得樟芝中的孢子萌发相关蛋白信息，最后用RT-qPCR技术对相关基因的转录水平进行分析。[结果]两组样品共有32个差异蛋白点，其中在24 h表达量上调的蛋白25个，下调的蛋白7个。将32个差异蛋白点挖取鉴定，成功鉴定24个。其中，与孢子萌发相关的蛋白有10个，分别为GerO、Ubc1、Cat-1、Snf1、Cas2、SfaD、Chaperonin、Fad5、Tyrosine-P和ChiA。[结论]该研究结果为进一步解析樟芝无性孢子萌发的分子机制提供了理论依据。

关键词:樟芝;孢子萌发;双向电泳;荧光定量PCR

Abstract:

[Objective] Germination-related proteins in Antrodia camphorata athroconidia were analyzed by two-dimensional gel electrophoresis (2DE), mass spectrum, and real time fluorescent quantitative PCR (RT-qPCR).[Methods] We used 2DE to analyze total proteins of Antrodia camphorata arthroconidia after 0 h and 24 h of incubation. We identified differential proteins by PDQuest software and MALDI-TOF-MS. Then, we obtained germination-related proteins in Antrodia camphorata arthroconidia by matching the amino acid sequences of identified proteins to a local protein database. Finally, we used RT-qPCR to quantify relative expression levels of germination-related genes.[Results] A total of 32 differential expressed proteins, of which 25 up-regulated and 7 down-regulated, existed between non-germinated (0 h) and germinated (24 h) arthroconidia. Among these differential proteins, 24 proteins were successfully identified, and 10 proteins were involved in arthroconidial germination including GerO, Ubc1, Cat-1, Snf1, Cas2, SfaD, Chaperonin, Fad5, Tyrosine-P, and ChiA.[Conclusion] The results provide a theoretical basis for understanding of molecular mechanisms of athroconial germination of Antrodia camphorata.

Key words:Antrodia camphorata;arthroconidial germination;2D gel electrophoresis;RT-qPCR