【作者】 任楠；

【导师】 潘力；

【作者基本信息】 华南理工大学 ， 微生物学， 2011， 硕士

【摘要】 丝状真菌具有良好的蛋白分泌能力,而且能对合成的真核蛋白正确地进行各种翻译后加工包括肽链的剪切和糖基化等,其糖基化修饰模式较酵母更类似于高等真核生物。由于在食品及食品加工中长期应用,许多菌种已被认定是安全的。与哺乳动物和昆虫等高等生物表达系统相比,丝状真菌培养方便、生长迅速、成本较低,表达的外源蛋白可以分泌到培养基中,下游的分离纯化简单。加之丝状真菌遗传转化系统研究的迅速发展,为在丝状真菌中表达外源蛋白奠定了基础。本文以泡盛曲霉为研究对象,拟通过根癌农杆菌介导建立其遗传转化体系。通过分泌蛋白图谱分析和药物敏感性分析,确定了以潮霉素B抗性基因和乙酰胺酶基因amdS作为筛选标记的宿主菌;基于Gateway克隆技术,构建了由高效启动子、终止子和目的基因表达框组成的根癌农杆菌双元载体,筛选标记为潮霉素B抗性基因和乙酰胺酶基因amdS;通过根癌农杆菌介导,将构建的双元载体转化泡盛曲霉,获得了含有目的基因的转化子,经过PCR验证、SDS-PAGE蛋白电泳和Western蛋白杂交证明目的基因已转化进入泡盛曲霉,表明根癌农杆菌介导泡盛曲霉转化具有可行性,所得转化子遗传稳定性高,适合作为异源蛋白的表达体系。更多还原

【Abstract】 Filamentous fungi could secrete a large amount of proteins, and they own many protein post-translational modification mechanisms such as peptide chain splicing, glycosylation and so on. Moreover, their glycosylation mode is much more similar with higher eukaryotes than yeast. Due to the long-term application in food industry, many species in filamentous fungi have been generally recognized as safe (GRAS). Compared to higher organisms such as mammalian and insect expression system, filamentous fungi are characteristic of simple nutrition, rapid growth, low costs and fast downstream separation and purification due to secreted expression. The great advances in fungal gene expression systems lay the foundation for genetic research in filamentous fungi.In this paper, transformation system for Aspergillus awamori was established, which was mediated by Agrobacterium tumefaciens. Based on the analysis of secreted protein expression profile and drug sensitivity, the resistant genes to hygromycin B and acetamidase (amdS) was determined as selection markers. Then, using the Gateway cloning technique, Agrobacterium tumefaciens binary vectors were constructed containing effective promoter, terminator and target gene ORF, with hygromycin B resistance gene and acetamidase gene amdS as selection markers. After transformation the binary vectors with Aspergillus awamori mediated by Agrobacterium tumefaciens, several transformants were obtained. And these transformants were verified by PCR amplication, SDS-PAGE and Western blotting, suggesting that target gene had been transformated into Aspergillus awamori. The above data proved that the Agrobacterium-mediated transformation of Aspergillus awamori was feasible, with high genetic stability of transformants. Therefore, Aspergillus awamori was suitable as heterologous protein expression system.更多还原