【作者】 陈美元；

【导师】 谢宝贵； 王泽生；

【作者基本信息】 福建农林大学 ， 微生物学， 2011， 博士

【摘要】 双孢蘑菇[Agaricus bisporus (J.E.Lange) Imbach]是世界性栽培与消费的食用菌,具有重要的经济价值和生态意义。As2796是我国多年来唯一的双孢蘑菇主栽品种,对其退化现象的分子机理研究具有重要的理论和实践意义。在本课题中,我们应用As2796正常菌株及其两个基质降解能力退化突变体菌株2796-3、2796-5作为比较研究的对象。主要研究内容和结果如下:(1)对双孢蘑菇2个基质降解能力退化菌株进行多代的无性繁殖(转管)试验及退化性状检测,确定了该退化现象可以稳定存在。对正常与退化菌株进行了混合接种试验,排除了病毒等微生物感染的因素,证明了该退化性状应是基因变异所引起的。进行了不同液体培养基的设计与培养,获得3个菌株在4种培养基中的12个菌丝体样品进行后续的研究。(2)对双孢蘑菇As2796及其分解基质能力退化菌株2796-3、2796-5的总DNA进行了153对引物组合的SRAP分析,结果未能发现两类菌株DNA水平的差异。根据试验结果推测,该退化变异的范围较窄,有可能是个别基因的突变,因而在DNA水平上难以发现。(3)应用同工酶聚丙烯酰胺凝胶电泳(PAGE)技术对4种不同液体培养基培养的双孢蘑菇As2796及其基质降解能力退化菌株2796-3、2796-5的菌丝体进行了分析,结果发现在部分培养基中正常与退化菌株的酯酶(EST)和多酚氧化酶(PPO)同工酶表型有明显差异,且2个退化菌株的同工酶表型变异一致,说明双孢蘑菇基质降解能力的退化与同工酶活性存在相关性。(4)对4种不同液体培养基培养的双孢蘑菇As2796及其基质降解能力退化菌株2796-3、2796-5进行了24对引物组合的mRNA差异显示(DDRT-PCR)分析,结果发现8对引物组合扩增出可重复的差异,并克隆了9条差异片段。序列分析结果显示9条差异片段代表了5个基因的差异,部分与不同生物的ATP结合盒式运输亚家族A某蛋白、碳水化合物酯酶家族4蛋白、几丁质脱乙酰基酶、乙酰木聚糖酯酶II及其它一些未知蛋白具有较高的同源性。(5)构建了双孢蘑菇As2796菌株的全长cDNA文库,文库初始库容达3.3x106 cfu/mL,插入片段长度范围约0.7-4.0Kb,平均长度约1.5Kb,重组率约82%,全长率约35%。文库扩增出了DDRT-PCR的5个差异基因全长编码序列(CDS),表明了该文库的有效性,今后可用于扩增所需的双孢蘑菇正常基因的全长cDNA或CDS。(6)以双孢蘑菇As2796及其2个退化菌株的总DNA为模板,扩增出了DDRT-PCR中5个差异片段所在基因带内含子的编码序列及其上游约1200bp调控序列并进行克隆与测序比较,结果表明这些基因与上游序列在三个菌株中并无实质性差异,据此推测退化性状更可能是调控蛋白基因变异所引起的。(7)对双孢蘑菇总蛋白质的双向电泳(2-DE或2D-PAGE)条件进行了摸索与优化,建立了双孢蘑菇蛋白质的2D-PAGE技术,获得了清晰的2D-PAGE图谱。结果表明双孢蘑菇的蛋白质大部分集中在分子量20-120KDa、等电点pI5.2-7.8的范围内。(8)对双孢蘑菇As2796及其基质降解能力退化菌株2796-3、2796-5进行了2D-PAGE分析,发现了2个明显的、可重复的差异蛋白质,在退化菌株中分别为上调和下调表达,且2个退化菌株表现一致。根据质谱(MALDI-TOF-MS)分析和数据库检索结果,2个差异蛋白质初步鉴定为肌动蛋白和NADH脱氢酶铁硫蛋白3,它们与细胞内信号传导或转录调控等过程关系密切。本研究首次发现了一些与双孢蘑菇基质降解能力退化相关的基因片段与蛋白质(酶),基于本研究结果提出双孢蘑菇基质降解能力退化分子机理是一个多级链式应答(逐级调控)过程的初步设想。即基质降解能力上的退化现象不是单一结构蛋白基因的变异,而可能是调控基因的变异,导致基因表达网络的改变,最终在转录水平、蛋白质水平、细胞水平及生物体上表现出系列差异,造成菌株在基质降解能力上的退化现象。该设想为进一步深入研究双孢蘑菇分解基质能力退化的分子机理奠定了良好的基础。更多还原

【Abstract】 Agaricus bisporus (J.E.Lange) Imbach is a kind of edible mushroom cultivated and consumed worldwide, and has high economic value and important ecological significance. As2796 is the only main variety of A.bisporus used in China during more than 10 years, so the study on the molecular mechanism of the degeneration characteristics of this variety has important theoretical and practical significance. In this subject, the normal strain As2796, as well as two substrate-decomposing ability degeneration mutants derived from which, 2796-3 and 2796-5, were used as two comparison groups. The main research contents and results are showed as below.(1) The 2 degeneration strains were transferred on PDA medium for dozens of generations, and their degeneration characters were comfirmed to be stable. The normal and abnormal strains were mixed and cultured togather, and the results exclued the factor of microorganisms infection, suggesting the degeneration to be caused by gene variation. Different liquid media were designed for strains culturing, and 12 mycelia samples of 3 strains in 4 kinds of liquid media were harvested for further study.(2) The technique of SRAP with 153 pairs of primers was used for the analysis of DNA differences between Agaricus bisporus As2796 and its mutants, 2796-3 and 2796-5. As a result, no difference was found between two kinds of strains. Based on the result, it was guessed that the variation might occur in few genes and was difficult to be found at DNA level.(3) The technique of isozyme PAGE was used for the analysis of the 3 strains cultured in 4 kinds of liquid media. The results showed that the esterase (EST) and polyphenol oxidase (PPO) isozyme phenotypes made obvious differences between the normal and degeneration strains in some kinds of media, and the differences occurred in both of the two degeneration strains, suggesting there to be relationship between the isozyme activity and the substrate-decomposing ability degeneration of A.bisporus.(4) The technique of mRNA differential display reverse transcription-PCR (DDRT-PCR) with 24 pairs of primers was used to analyze mRNA differences between the wild type of A. bisporus strain As2796 and its substrate-decomposing ability degeneration strains 2796-3 and 2796-5 cultured in 4 kinds of different liquid media. As a result, 8 pairs of primers showed repeatable differences, and 9 special fragments were cloned. Sequencing analysis showed that 9 special fragments represented 5 different genes. Some of them have high homologies with an ATP-binding cassette transporter subfamily A protein, carbohydrate esterase family 4 protein, chitin deacetylase, acetyl xylan esterase II and some other hypothetical proteins of several kinds of organisms.(5) The full-length cDNA library of A.bisporus As2796 was constructed. The library had a content of 3.3×106 cfu/mL, an insertion range of about 0.7-4.0Kb, an average cDNA length of 1.5Kb, a recombination efficience of about 82%, and full-length ratio of about 35%. The coding sequences (CDS) of 5 different genes found in DDRT-PCR were got from the library by PCR amplification, which proved the validity of the library for amplifying full-length cDNA or CDS of A.bisporus genes in need.(6) Using the total DNAs of A.bisporus As2796 and 2 degeneration strains as templates, the CDS with introns and about 1200bp upstream regulative sequences of 5 different genes found in DDRT-PCR were amplified, cloned and sequenced. The comparison of the sequences among 3 strains showed that the genes and upstream sequences have no repeatable difference, so the degeneration might be caused by the mutation of regulative protein genes.(7) The conditions for A.bisporus proteome two-dimensional electrophoresis (2-DE or 2D-PAGE) were studied and optimized, and the proteome 2D-PAGE technique of A.bisporus was built up, resulting in clear 2D-PAGE patterns. It was showed that most of the proteins of A.bisporus focus in the range of molecular weight (MW) 20-120KDa and isoelectric point (pI) 5.2-7.8.(8) The technique of 2D-PAGE was used to analyze the protein expression differences between the wild type Agaricus bisporus As2796 and its substrate-decomposing ability degenerated strains 2796-3, 2796-5. As a result, two proteins were found to be up- and down-expressed, respectively, with obvious and repeatable profiles in both degenerated strains when grown in a liquid culture. Based on the results of MALDI-TOF-MS analysis and database searching, these two proteins were identified as an actin and a putative NADH dehydrogenase iron-sulfur protein 3, respectively, and both of which had close relationship with intracellular signal conduction or transcription regulation.In this work we found some gene fragments and proteins (enzymes) related to the substrate- decomposing ability degeneration of A.bisporus for the first time. Based on our study, the molecular mechanism of substrate-decomposing ability degeneration in A.bisporus was preliminarily supposed to be a course of gradual chain regulation, i.e. the degeneration phenotype was not caused by the variation of some certain structural protein genes, but the mutation of regulative protein genes, which changed the gene expression network, resulting the series of differences at mRNA, protein, cell and organism level, and finally brought the degeneration of substrate-decomposing ability. This supposition supported a good basic for the further research on the molecular mechanism of substrate-decomposing ability degeneration of A.bisporus.更多还原