【作者】 陆兆明；

【导师】 宋思扬；

【作者基本信息】 厦门大学 ， 微生物学， 2008， 硕士

【摘要】 双孢蘑菇[Agaricus bisporus Lange(Imbach)]是目前世界上人工栽培最广泛、产量最高、消费量最大的食用菌。它属于稳温结实性的菌类,温度是影响其生长的主要因素。加强和深化双孢蘑菇的分子生物学研究,特别是充分运用现代生物技术研究双孢蘑菇的热胁迫蛋白质表达差异以及耐热分子机理,对培育耐温、高产、优质的菌株具有十分重要的理论和实践意义。首先,本研究对双孢蘑菇耐温型02菌株和温度敏感型8213菌株进行ITS鉴定。经过ITS鉴定分析,02和8213菌株是双孢蘑菇种内的不同菌株。将02和8213菌株各分为4组:24℃常温培养、先32℃热诱导6h,再37℃热处理6h、37℃热处理12h和42℃热处理12h。观察不同温度处理条件下菌丝体的形态变化,结果发现:02菌株比8213菌株耐高温;较低的亚致死诱导温度对于提高热耐受能力是有帮助的。其次,通过双向电泳联用质谱技术研究02菌株热处理前后总蛋白2-DE图谱的变化以及同时热处理的02和8213菌株的总蛋白2-DE图谱差异,并对差异表达的蛋白质点进行MALDI-TOF-MS分析。2-DE图谱分析结果表明:热处理后的02菌株,有32个蛋白质斑点出现明显的差异,其中热诱导出现蛋白质5个,表达量上调蛋白质14个,抑制表达蛋白质4个,表达量下调蛋白质9个;02和8213菌株同时热处理后,有41个蛋白质斑点有较为明显的差异,其中18个蛋白质为02菌株独有,17个蛋白质在02菌株中表达上调;3个蛋白质为8213菌株独有,3个蛋白质在8213菌株中表达上调。应用MALDI-TOF-MS分析73个差异蛋白质点,在MASCOT数据库中检索比对,共鉴定出58个差异表达蛋白质。将这58个已鉴定差异蛋白质序列在COGs数据库中进行比对功能分类,可分为以下几类:分子伴侣类、能量代谢类、物质代谢类、细胞过程和信号传导类、遗传信息类及功能未知蛋白质。功能簇中分子伴侣蛋白质共有6个,占全部已鉴定差异蛋白质的10.3%,这表明包括热激蛋白在内的分子伴侣蛋白质对于耐高温可能起重要的作用。与能量代谢和物质代谢相关的蛋白质有14个,占全部已鉴定差异蛋白质的24.1%,说明在热处理中双孢蘑菇的正常的能量和物质代谢受到了干扰。细胞信号、遗传信息传递相关蛋白质共有11个,占已鉴定差异蛋白质的19%,说明双孢蘑菇在基因转录水平上对热激反应进行调控。在差异蛋白质中,有两个被鉴定为Heat shock protein HSS1和HSP70。HSS1在02菌株热处理后表达上调;在02与8213菌株同时热处理的蛋白质组比较中,HSP70在02菌株蛋白质组中表达量较大。我们通过real-time PCR检测,发现02菌株热处理后HSS1和HSP70基因的表达量都上升5倍以上;同样进行热处理的02和8213菌株中,HSS1和HSP70基因在02菌株中的表达量分别为8213菌株的1.6倍和2.8倍,说明基因转录水平上调引起了HSS1和HSP70表达量的提高。本研究通过双向电泳技术手段找到一些耐温02菌株热处理前后的差异表达蛋白质以及耐温02菌株和温度敏感型8213菌株同时热处理的差异表达蛋白质,并通过MALDI-TOF-MS和肽指纹质量图谱鉴定了部分差异蛋白质。通过分析已鉴定差异蛋白质的功能,初步了解双孢蘑菇可能的热应激机制,这将有助于揭示双孢蘑菇的耐热分子机理,为基因工程手段培育耐高温双孢蘑菇菌株提供理论基础。更多还原

【Abstract】 Agaricus bisporus is the manual cultivation most widespread,the output highest, the consumption quantity biggest edible fungus in the present world.It belongs to stable-temperature fruitfulness fungi,temperature is the main factor affecting its growth.The enforced and deeply research of thermotolerance moleculer biology of A.bisporus,especially researching on different expression proteins of heat shock and heat-resistant molecular mechanism by the modern biological technology,is important to breeding of A.bisporus for heat-tolerance,high yield,high quality in theory and practice.First,the ITS test are carried out to identify strains 02 and 8213.strains 02 and 8213 are identified to different strains of A.bisporus by ITS test.Strains 02 and 8213 are grouped into four groups:cultured on 24℃;cultured on 32℃for 6h,then cultured on 37℃for 6h;cultured on 37℃;cultured on 42℃.From the morphotype of A.bisporus in different temperature conditions we found:strain 02 is more thermotolerance than strain 8213;lower sublethal temperature is helpful to thermotolerance.Secondly,We have used two dimensional electrophoresis(2-DE) and Mass spectrometry(MALDI-TOF/MS) to identify proteins that are differentially expressed in group of strain 02 under heat stress and under normal temperature and group of strains 02 and 8213 under heat stress.From 2-DE data of strain 02 under heat stress and under normal temperature,32 resolved differentially expressed proteins were detected,5 of those were induced,14 of those were up-regulated,4 of those disappear,9 of those were down-regulated.From 2-DE data of strains 02 and 8213 under heat stress,41 resolved differentially expressed proteins were detected,18 of those were only found in strain 02,17 of those were were up-regulated in strain 02,3 of those were only found in strain 8213,3 of those were up-regulated in strain 8213.All the 73 spots were subjected to tryptic digestion followed by MALDI-TOF-MS, compared in Mascot.Total 58 proteins were identified and classified into different functional categories by COGs.They are classified into molecular chaperon,energy, metabolism,cellular processes and signaling,information storage and processing, function unknown.Six of these are molecular chaperon proteins,accounting for 10.3%, showed that molecular chaperon proteins(including HSP) are helpful to thermotolerance.Fourteen of these are energy and metabolism proteins,accounting for 24.1%,showed that the normal energy metabolism is interfered.Eleven of these are cellular processes and signaling,information storage and processing proteins, accounting for 19%,showed that A.bisporus regulate heat shock response on gene transcription level.Heat shock protein HSS1 is identified up-regulated in strain 02 under heat stress. HSP70 is identified up-regulated in strain 02 caompared to strain 8213.From real-time PCR detection,we find HSS1 and HSP70 are up-regulated to 5 times;in strains 02 and 8213 under heat stress,HSS1 and HSP70 are up-regulated to 1.6 and 2.8 in strain 02,showed that HSS1 and HSP70 are up-regulated induced by gene transcription level.The study found differentially expressed proteins in group of strain 02 under heat stress and under normal temperature and group of strains 02 and 8213 under heat stress by 2-DE,then identify them by MALDI-TOF-MS and PMF.Analyze the identified differentially expressed proteins and understand the mechanism of heat stress is helpful to reveal heat-resistant molecular mechanism of A.bisporus and breed heat-tolerance strain by genetic engineering.更多还原