【作者】 张成省；

【导师】 李多川；

【作者基本信息】 山东农业大学 ， 植物病理学， 2002， 硕士

【摘要】 在各种不同的生命现象中，识别是中心事件，也是许多基于细胞间互作的生命进程中的第一步。真菌细胞壁作为细胞的最外层部分，调节着真菌(包括菌寄生真菌或寄主真菌)与周围环境最初的物理化学相互作用，细胞壁结合蛋白质在这些过程中扮演着重要角色，细胞壁蛋白质的研究成为生物间相互识别机制研究的热点。菌寄生是菌寄生真菌与寄主真菌间的一种寄生方式，结合于寄主真菌菌丝细胞壁上的特异性蛋白—凝集素被认为是菌寄生过程中的重要识别因子。 纤细齿梗孢Olpitrichum tenellum系接触性活体营养菌寄生真菌，寄主之一是Alternaria alternata，关于这一类菌寄生真菌与寄主真菌间的识别机制研究国内外尚未见报道。本研究选择二者作为研究系统，分离纯化了寄主细胞表面的特异性蛋白质凝集素，对其在菌寄生过程中的作用作了初步探讨，为进一步研究菌寄生过程中的信号传递奠定基础，为阐明生物间的相互作用机理提供一定证据；同时对真菌菌丝细胞壁可溶性蛋白的分离方法也作了研究，为真菌菌丝细胞壁研究提供方法参考和实验依据。结果如下： 1．以A．alternata为研究材料，分别用三种不同的化学方法，对真菌菌丝细胞壁蛋白质的抽提方法进行了比较和研究。结果表明依次经过液氮研磨、超声波细胞粉碎机和细胞匀浆混合器处理，可使细胞壁破碎率≥99％在一定条件下(1200g，2min)离心即可制备较纯的细胞壁，纯度约95％。将分离到的细胞壁分别用三种不同的化学方法即碱抽提(常温)、热碱抽提和SDS抽提，并对抽提液的蛋白含量及组分进行分析，结果表明三种方法均为有效的抽提A．alternata菌丝细胞壁蛋白的方法。其中，热碱法抽提蛋白含量最大，常温碱法次之，SDS法最小；三种方法所抽提蛋白主要成分均一致(≥40％)，但所抽提的蛋白组分又有差异。SDS法和常温碱法相似性极大(≥80％)，而二者与热碱法差异相对较大；三种方法所得到的细胞壁抽提液的含糖量各不相同，常温碱法含糖量很低，SDS法与热碱法含糖量则很高，以SDS法为最高：含糖量越高，其对蛋白质研究影响越大。本研究仅仅对蛋白质的抽提方法做了比较和研究，这些方法所抽提到的非蛋白组分对随后的研究是否有影响或有哪些影响还需进一步的研究。 2．A．alternata菌丝细胞壁可溶性蛋白抽提液对O．tenellum的孢子具有较强的凝集力，用氯仿一异戊醇去除蛋白后，对O．tenellum的孢子的凝集力则显著下降，这说明起凝集作用的因子很可能是一种蛋白质。经硫酸铵沉淀，DEAE-Sepharose阴离子交换柱层析和Sephacryl S-100分子筛层析分离到了这种蛋白，活性分析表明它是一种凝集素，定名为AAL，分子量为37kDa，其等电点是pH4.7，最大紫外吸收波长为282nm。经糖染色鉴定为一种糖蛋白，中性糖含量约为14％。本研究结果表明AAL在A．alternata菌丝和O．tenellum的孢子吸附事件中扮演着重要角色，对O．tenellum的孢子具有极强的凝集力，当其缺乏时，孢子凝集力显著下降，暗示AAL很可能起识别因子的作用，与菌寄生真菌细胞表面的特殊位点(可能为糖蛋白)相 张成省：Alternaria alternate菌丝纫胞壁凝集素的分离纯化及特性研究结合，以达到相互识别和附着的作用。更多还原

【Abstract】 Recognition is a central event in a variety of biological phynomena and the first step in numerous processes based on cell-cell interactions. As the most external part of the cell, cell wall mediates the initial physical and chemical interaction between microorganisms and the environment, including the host. Proteins have been implicated in most of cell wall functions.Recent researches have been limited primarily to the function of proteins as adhesions, and results showed such recognition may be mediated by lectin.In recent studies, great attention has been paid to interaction between mycoparasites and mycohost. Olpitrichum tenellum is a biotrophic contact mycoparasites, Alternaria alternata is one of its host fungi. The recognition mechenism between them was by far not reported. This research focused on the purification, properties and adhesion activity of a lectin from A.alternata in order to reveal the recognition mechanism between mycoparasite and mycohost, also to provide basis for further research.There has been little comparison of the various methods used to extract the cell wall proteins. In the first part of this article, three extracts for A.alternata isolated mycelia cell walls were compared: (i) cold alkaline extract;(ii) SDS extract;(iii) hot alkaline extract. The extracts were examined by thin layer scanning. Results showed the choice of extracting reagents and techniques,as well as the sequence of extraction methods may affect both qualitative and quantitative solubolization of cell wall proteins.In the second part of this article, a lectin from isolated A.alternata cell walls was purified to SDS-PAGE homogeneity by ammonium sulfate fraction, DEAE-Sepharose Fast Flow chromatography and Sephacryl S-100 chromato-graphy. The lectin, named AAL, was a glycoprotein with an apparent molecular weight of 36.3 kDa and 37.2 kDa by SDS-PAGE and gel filtraction,respectively. AAL may be a cell recognition molecule which mediates the adhesion of O.tenellum cells to A.alternata cells. Adhesion experiments between AAL and conidia of O.tenellum , >90% conidia adhered within 30 min at concentrations as low as 3.325 ug ml"1. Compared to adhesion in the absence of AAL, there was no significant effect on spore adhesion.更多还原