iTRAQ-based comparative proteome analyses of different growth stages revealing the regulatory role of reactive oxygen species in the fruiting body development of Ophiocordyceps sinensis.
iTRAQ-based comparative proteome analyses of different growth stages revealing the regulatory role of reactive oxygen species in the fruiting body development of Ophiocordyceps sinensis.
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基于iTRAQ的不同生长阶段的比较蛋白质组分析揭示活性氧在冬虫夏草子实体发育中的调节作用
作者：
Tong X;Wang F;Zhang H;Bai J;Dong Q;Yue P;Jiang X;Li X;Wang L;Guo J
本研究采用等压相对和绝对定量标记(ITRAQ)技术，结合LC-MS/MS和生物信息学，对冬虫夏草子实体发育的三个关键阶段--菌核(ST)、原基(PR)和成熟子实体(MF)的蛋白质组进行了分析，重点研究了子实体发育相关的蛋白质及其可能的发育机制。共鉴定出1,875种蛋白质。主成分分析(PCA)表明PR和MF之间的蛋白质图谱比ST更相似。差异积累蛋白(DAPs)分析显示，ST与PR、PR与MF、ST与MF的差异积累蛋白分别为510、173、514个。共有62个共享的DAP被鉴定出来，主要富含与“碳运输和机制”、“对氧化应激的反应”、“抗氧化活性”和“翻译”相关的蛋白质。KEGG和GO数据库显示，DAP在“初级代谢(氨基酸/脂肪酸/能量代谢)”、“对氧化应激的反应”和“过氧化物酶”方面含量丰富。此外，34个参与ROS代谢的DAP被识别，并使用hClusterR程序包中实现的层次聚类在三个阶段进行聚类。有人建议，它们的作用和基本机制可能因阶段而异。ROS可能在ST真菌致病、PR中的子实体启动、MF中的有性繁殖和高原适应中起作用。鉴定了与ROS相关的关键蛋白，如超氧化物歧化酶(SOD，T5A6F1)、NOR-1(T5AFX3)、电子传递蛋白(T5AHD1)、组氨酸磷酸转移酶(HPT，T5A9Z5)和谷胱甘肽过氧化物酶(T5A9V1)。此外，用2，7-二氯荧光素二乙酸酯(DCFH-DA)比色法测定三个阶段的ROS积累量。与PR和ST期相比，MF期的ROS积累要强得多。用DCFH-DA对肉芽分生孢子和子实体切片进行染色，在荧光显微镜下观察到ROS分布在分生孢子和子囊内。此外，与ST期和PR期相比，MF的SOD活性在三个阶段都有所增加，而CAT活性的增加幅度更大。这表明ROS可能以梯度依赖的方式调节子实体的发育。用实时定量聚合酶链式反应(qRT-PCR)在mRNA水平上分析了6个DAP的编码区序列。结果支持DAPS分析结果和蛋白质组测序数据。我们的发现为了解中华稻曲霉子实体发育和高原适应的生物学提供了蛋白质组学的视角，为大产业开发这一有价值的真菌提供了信息。
In this study, using an isobaric tags for relative and absolute quantitation (iTRAQ ) approach coupled with LC-MS / MS and bioinformatics, the proteomes were analyzed for the crucial three stages covering the fruiting body development of Ophiocordyceps sinensis, including sclerotium (ST), primordium (PR) and mature fruiting body (MF), with a focus on fruiting body development-related proteins and the potential mechanisms of the development. A total of 1,875 proteins were identified. Principal Component Analysis (PCA) demonstrated that the protein patterns between PR and MF were more similar than ST. Differentially accumulated proteins (DAPs) analysis showed that there were 510, 173 and 514 DAPs in the comparisons of ST vs. PR, PR vs. MF and ST vs. MF, respectively. A total of 62 shared DAPs were identified and primarily enriched in proteins related to ‘carbon transport and mechanism’, ‘the response to oxidative stress’, ‘antioxidative activity’ and ‘translation’. KEGG and GO databases showed that the DAPs were enriched in terms of ‘primary metabolisms (amino acid/fatty acid/energy metabolism)’, ‘the response to oxidative stress’ and ‘peroxidase’. Furthermore, 34 DAPs involved in reactive oxygen species (ROS) metabolism were identified and clustered across the three stages using hierarchical clustering implemented in hCluster R package . It was suggested that their roles and the underlying mechanisms may be stage-specific. ROS may play a role in fungal pathogenicity in ST, the fruit-body initiation in PR, sexual reproduction and highland adaptation in MF. Crucial ROS-related proteins were identified, such as superoxide dismutase (SOD, T5A6F1), Nor-1 (T5AFX3), electron transport protein (T5AHD1), histidine phosphotransferase (HPt, T5A9Z5) and Glutathione peroxidase (T5A9V1). Besides, the accumulation of ROS at the three stages were assayed using 2,7-dichlorofuorescin diacetate (DCFH-DA) stanning. A much stronger ROS accumulation was detected at the stage MF, compared to the stages of PR and ST. Sections of ST and fruit-body part of MF were stained by DCFH-DA and observed under the fluorescencemicroscope, showing ROS was distributed within the conidiospore and ascus. Besides, SOD activity increased across the three stages, while CAT activity has a strong increasement in MF compared to the stages of ST and PR. It was suggested that ROS may act in gradient-dependent manner to regulate the fruiting body development. The coding region sequences of six DAPs were analyzed at mRNA level by quantitative real-time PCR (qRT-PCR). The results support the result of DAPs analysis and the proteome sequencing data. Our findings offer the perspective of proteome to understand the biology of fruiting body development and highland adaptation in O. sinensis, which would inform the big industry of this valuable fungus.
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DOI：
10.1155/2012/791963
发表时间：
2012
期刊：
Journal of signal transduction
影响因子：
--
作者：
Belozerskaya TA;Gessler NN;Isakova EP;Deryabina YI
通讯作者：
Deryabina YI
影响因子：
16.5
作者：
Krijgsheld P;Bleichrodt R;van Veluw GJ;Wang F;Müller WH;Dijksterhuis J;Wösten HA
通讯作者：
Wösten HA
DOI：
10.1615/intjmedmushr.v10.i3.30
发表时间：
2008-01-01
影响因子：
1.2
作者：
Holliday, John;Cleaver, Matt
通讯作者：
Cleaver, Matt
影响因子：
3.9
作者：
Díaz, A;Muñoz-Clares, RA;Hansberg, W
通讯作者：
Hansberg, W
影响因子：
4.8
作者：
Bang, Ye-Ji;Oh, Man Hwan;Choi, Sang Ho
通讯作者：
Choi, Sang Ho