猪苓菌丝形成菌核差异蛋白质组学研究

Differential proteome analysis of sclerotium formation stages of Polyporus umbellatus

药用真菌猪苓菌丝形成菌核分子机制一直是人们关注的热点问题。本研究首次对猪苓进行了系统的蛋白质组学研究。基于液质联用技术并在数据依赖性采集模式下,成功鉴定了猪苓菌丝形成菌核的初始期、菌核生长期和成熟期的蛋白质组,共鉴定蛋白1 391个(global FDR 1%)。采用先进的SWATH MSALL的非标记定量方式对初始期菌丝和菌核进行了差异蛋白质组学研究,从中定量蛋白1 234个,占鉴定蛋白数量的88.7%。猪苓菌丝形成菌核过程中有378个蛋白差异表达。差异蛋白GO注释结果表明,在猪苓菌丝形成菌核过程中,差异蛋白参与的蛋白功能和生物学过程多样,以催化和结合为主,还参与感知环境刺激、信号转导、电子转移、过氧化活性、菌丝生长及胞壁合成等功能。进一步推测分析表明,猪苓菌丝形成菌核可能是由于缺氧或温差等多种环境胁迫诱导所致,生成活性氧并形成氧化应激状态。信号通过丝裂原活化蛋白激酶通路(MAPK通路)途径向下游传递,可能调控了胞壁蛋白糖基化修饰,进而促进猪苓菌丝极性生长和菌丝形态学变化而形成菌核。KEGG代谢通路分析表明,差异蛋白还主要参与了糖、脂质和氨基酸等初级产物以及甾醇、多聚乙酰等次级代谢物的合成与代谢。因此,对差异蛋白质组的深入解析和研究有利于揭示猪苓菌丝形成菌核的蛋白分子机制,具有较强的理论和现实意义。

The mechanisms of sclerotial formation from the hyphae of the medicinal fungus Polyporus umbellatus was investigated. Proteomics based on LC-MS was introduced into P. umbellatus. A total of 1 391 proteins were identified from sclerotia and hyphae at initial, developmental and mature phases based on data dependent acquisition（DDA） model. In total, 1 234 proteins were quantified from initial sclerotia and hyphae based on SWATH（Sequential Window Acquisition of all theoretical fragment ions） label-free model, and there were 378 proteins expressed differentially including 322 known and 56 unknown proteins. GO analytic results showed that these differential proteins were of varieties in molecular function and biological process during the sclerotial formation from the hyphae. Most of differential proteins were involved in catalytic and binding activities, and some were associated with response to stimulus, signal transduction, electron carrier, peroxidase activity, filamentous growth, cell wall biogenesis and so on. Further analysis results showed that types of stress（such as difference in temperature, hypoxia, etc.） induced the sclerotial formation of P. umbellatus from hypha, resulted in reactive oxygen species（ROS） generation and then developed into oxidative stress（OS）. The signal transduced via MAPK pathway, and might regulate glycosylation of cell wall proteins, which were associated with cell polarities and filamentous hypha morphological changes. Besides, KEGG analysis indicated that proteins involved in synthesis and metabolism of secondary metabolites（such as terpenoids and polyketides） expressed differentially. It was the first time study on the proteome of P. umbellatus, which may be useful to deepen the knowledge on sclerotial formation.