瑞氏木霉pyrG基因缺陷型菌株筛选以及GnTI和VHb基因表达研究

【作者】 龙昊；

【导师】 汪天虹；

【作者基本信息】 山东大学 ， 遗传学， 2006， 硕士

【摘要】 丝状真菌作为微生物具有结构简单、生长迅速、操作简便的优点，同时作为低等真核生物，又具有真核基因表达机器和真核蛋白质翻译后修饰加工装置，具有与哺乳动物系统相似的蛋白修饰性能。在发酵工业中，丝状真菌通常用于大规模生产酶制剂、有机酸等产物，其中有些菌株胞外酶产量可达20～40g/L。因此近年来丝状真菌分子生物学逐渐成为现代分子生物学研究的一个热点。最近已开始对丝状真菌的糖基化途径进行改造，以期得到能够产生与哺乳动物糖基化形式相近的工程菌株。 瑞氏木霉(Trichoderma reesei)是一种无性繁殖的丝状真菌，通常用于纤维素酶、半纤维素酶的生产。分子生物学证据显示瑞氏木霉是子囊菌Hyporcea jecorina的无性繁殖系。瑞氏木霉具有典型的真核蛋白修饰特性和理想的蛋白分泌能力。近年来，瑞氏木霉已作为基因工程宿主菌用于生产外源真核蛋白，并且为了生产药用人源蛋白而进行遗传改造，如其N-糖基化路径的人源化改造。 为了利用pyrG基因选择标记进行T．reesei转化，本文工作首先利用紫外线诱变技术获得了一株T．reesei pyrG基因缺陷菌株M23。M23在不含尿嘧啶核苷酸的培养基上无法生长，而转化含有pyrG基因的质粒后便可以在无尿嘧啶核苷酸的培养基上生长。转化率约为每微克质粒200～300个转化子。 本文利用T．reesei cbhl启动子和终止子构建了人N-乙酰氨基葡萄糖转移酶Ⅰ基因的真菌表达载体pCGNT，然后将在构巢曲霉trpC启动子和终止子控制下的透明颤菌血红蛋白(VHb)表达盒插入其中，构建了表达载体pUCGV。 用pUCGV质粒与pAN7-1质粒共转化T．reesei菌株U4，从含有150 mg/L潮霉素的平板上挑出约70个转化子。同时用pUCGV质粒与pAB4-1质粒共转化T．reesei菌株M23，从不含Uridine的平板上挑出约60个转化子。 PCR进行分子验证，得到一株GnTI和VHb基因稳定遗传的转化子T108。Southren Blot分析显示GnTI和VHb基因整合到T108染色体上。RT-PCR和SDS-PAGE分析表明GnTI和VHb基因在T108中已经转录并翻译。虽然使用CO差光谱法检测VHb的活性结果不明显，但是生长曲线显示在低氧条件下T108的生长速度高于出发菌株M23约10％。更多还原

【Abstract】 Filamentous fungus has the advantages of simple structure and fast growth, so it is easy to manipulate and culture. Being a kind of lower eukaryotes, it also has eukaryotic protein modification apparatus and the post translation modification mechanism, which is similar to that of mammals. Filamentous fungi are commonly used in fermentation industry for large scale production of enzmys and organic acids, several of these enzymes can be produced up to 20～40g per liter. So molecular biological research on filamentous fungus has been one of the hotspots. A start has been made to engineer the glycosylation path way in filamentous fungi to obtain stains that show a more mammalian-like type of glycosylation.Trichoderma reesei is an asexually reproducing filamentous fungus, mainly used in cellulase, hemicellualse production. The molecular evidence shows that T. reesei is an asexual, clonal line derivative of the ascomycete Hyporcea jecorina. Trichoderma reesei has the typical eukaryotic character of protein modification as well as the ideal capability of secreting proteins. Recently, T. reesei has been used in production of heterologous eukaryotic proteins as a host stain and genetic modification has been made for the therapeutic proteins production, such as the humanization of N-glycosylation pathways.In order to use the pyrG gene select marker in the transformation of T. reesei, first we obtained a T. reesei pyrG gene deficient strain M23 by using the UV mutation technology. The strain M23 cannot survive on medium without uridine until it is transformated with the vector of the pyrG gene. The transformation frequency is approximate 200～300 transformants per μg plasmids.In this study, the fungal express vector pCGNT used to express the human N-acetylglucosaminyltransferase I was constructed with the promoter and terminator of T. reesei cbh1. After that, the expression cassette of Vitreoscilla Hemoglobin (VHb) contains the promoter and terminator of A. nidulans trpC was inserted into pCGNT to更多还原