【作者】 李军辉；

【导师】 潘迎捷； 冯志勇；

【作者基本信息】 南京农业大学 ， 微生物学， 2006， 硕士

【摘要】 香菇(Lentinula edodes(Berk.)Pegler)是著名的大型木腐真菌，在我国有已有800多年的栽培历史。香菇子实体的形成是香菇生活史中最复杂的发育过程，需要遗传、环境、生理等因素的协同作用。在香菇子实体发育的起始阶段中，菌丝首先在营养逆境的胁迫下进行局部地聚集而形成扭结体，随后扭结体的密度逐渐增加而形成致密菌丝团，致密菌丝团最终发育为原基，原基本质上是子实体的胚胎结构。本文建立了适合香菇不同发育阶段组织的RNA高效提取方法，与其他常规RNA提取方法相比，该方法的独特之处在于用有机溶剂抽提之前，加入了饱和NaCl溶液，从而促进了多糖物质与蛋白质的去除，并提高了RNA的产量，为下游的分子生物学操作打下了基础。另外，本文运用mRNA差异显示技术比较了香菇菌丝体、菌丝扭结体、致密菌丝团以及原基等四个发育阶段的基因表达差异。四个发育阶段的组织提取RNA后进行反转录，之后用随机引物与锚定引物组合对cDNA样品进行扩增，再用琼脂糖凝胶电泳进行差异条带的展示。结果表明四个发育阶段的基因表达差异不甚明显，最终挑选了17个差异表达的ESTs基因片段进行克隆测序。通过与Gene Bank数据库的比对，发现这些ESTs片段涉及蛋白质的合成、细胞壁的修饰、菌丝间的相互作用、碳氮源的代谢、物质的运输、信号传导、细胞的复苏等过程。其中，一个差异片段被证实为内切多聚半乳糖醛酸酶基因，通过Real-time PCR定量分析，发现该基因在疏松菌丝体、菌丝扭结体和致密菌丝团等三个阶段的表达相对较低，而在原基发育阶段呈现大量的表达，表明该基因可能与香菇原基的形态发生具有密切的关系。更多还原

【Abstract】 Lentinula edodes (Berk.) Pegler is a kind of well-known large wood decaying fungus,with a cultivation history of more than 800 years in China.The fruiting body formation of L. edodes is the most complex developmental processin the life cycle of L. edodes. It is a highly organized process, which requires thecoordination between genetic, environmental, and physiological factors. In the initial stageof fruiting body development, upon nutritional depletion, hyphae form localized, highlybranched structures termed hyphal knots. Hyphal knots then develop into globosestructures, called sclerotia. The hyphae of the sclerotium differentiate further to formprimordium, that is essentially an embryonic fruiting body. This paper presents a simpleand inexpensive procedure for RNA extraction from L. edodes at various developmentalstages, especially from recalcitrant tissues of the fruiting body. When compared with otherroutine methods for RNA purification, the unique characteristic of this procedure isinclusion of a saturated NaCl solution treatment before continuing with organic solventextraction. This step promotes the removal of polysaccharides and proteins, and high yieldsof RNA can be readily obtained. Then the technique of mRNA differential display is usedto investigate the difference of gene expression between mycelium, hyphal knots, sclerotiaand primordium. Total RNA is firstly extracted from tissues at four different developmentalstages mentioned above, then cDNA is synthesized and RT-PCR performed using anchorprimers and arbitrary primers. After electrophoresis in agar gel, DNA fragments aredifferentially displayed. On the whole, the difference of gene expression betweenmycelium, hyphal knots, sclerotia and primordium is not obvious. By homology alignmentwith the database in Gene Bank, seventeen ESTs with altered expression involved inprotein synthesis, modification of cell wall, hyphal interaction, metabolism of C and Nsources, substrate transport, signal tranduction and cell rehabilitation are obtained. One ofthese ESTs proves to be the endopolygalacturonase gene, which shows an abundant expression pattern at the primordium stage after confirmation by Real-time PCR, while itsexpression at the three preceding stages is relatively low, demonstrating that it may beclosely invovled in primordim morphogenesis.更多还原