【作者】 王志耘；

【导师】 陶美凤；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 随着天蓝色链霉菌A3(2)和阿维链霉菌MA-4680的全基因组序列的发表，链霉菌的基础研究已进入后基因组时代，大量新基因和功能未知基因的功能有待鉴定。大规模突变体库的构建是功能基因组研究的一个重要内容，而利用转座子进行插入突变构建突变体库则是目前广泛应用的方法之一。 转座子是基因功能研究十分有用的工具。但目前在链霉菌中应用的大多数转座子却普遍存在转座效率低下或操作繁琐等缺点。仅有Tn4556及其衍生物和Tn5衍生转座子能够被应用于突变体库的构建和突变菌株的筛选。但是它们依然存在各种缺陷。 针对现有转座子系统的一些缺陷，构建了应用于链霉菌中的携带微型转座子mini-Tn4560A的质粒载体pHL265。它携带的转座酶基因tnpA位于转座子mini-Tn4560A外部，转座发生后，质粒载体pHL265由于不具有链霉菌复制子而不能在宿主菌中存在，失去了转座酶基因的微型转座子理论上将不可能发生二次转座；带有大肠杆菌与链霉菌进行属间接合转移的起始位点oriT，将有利于转座子载体通过接合转移的方式从大肠杆菌导入到链霉菌中，大大降低了原生质体转化过程中自体突变的可能；转座子直接进行体内转座，省去用Tn5体外转座时要构建基因组文库，再将体外转座突变体库重转入链霉菌，筛选突变菌株的复杂过程。 利用该转座子系统转座筛选得到天蓝色链霉菌M145的约1000株转座突变株。其中不乏表型异常的菌株。选取其中的8株，经Southern杂交验证可知该转座子的转座基本具有随机性；通过连续的松弛传代培养可知该转座子在宿主DNA中稳定存在。此系统为链霉菌功能基因组的研究提供了一个新的技术手段。更多还原

【Abstract】 The basic researd on Streptomycestes enters the post-genomics era since the published of Streptomyces coelicolor A3 (2) and Streptomyces avermitilis MA-4680 genome. Function of many novel genes or unknown genes is to be identified. Construction and screening of mutant library is an important strategy in functional genomics, and transposon is a useful tool to construct mutant libraries.Transposon is a very useful tool of gene function analysis. However, currently utilized transposons in Streptomyces have many disadvantages. Such as low transposition frequency or complicated to use. Some currently employed transposons (Tn4556 and Tn5 derivatives) had been used to construct mutant libraries, but they are not perfect at all.Aiming at improving currently used transposons, a novel transposon plasmid pHL265 was constructed containing a mini-transposon mini-Tn4560A. The pHL265 has several advantages. First, it a transposase gene tnpA located beyond the mini-transposon mini-Tn4560A. It contains no replication origin region for replication in Streptomyces. In theory a transposition mutagenized strain will not be subject to a second transposition because tnpA will be lost during the first transposition process. Scecond, pHL265 also contains oriT, the origin of transfer from RK2, which can be used to introduce DNA from E. coli to Streptomyces by conjugation. Conjugation will minimize mutation usually occurred in protoplast transformation. Finally, in-vivo transposition using pHL265 was much more concisely or directly, compared to in-vitro Tn5 transposition in which construction of a cosmid genomic library is required and the transposition mutagenized cosmid library has to be reintroduced into Streptomyces and screen for mutants.About one thousand transposition mutants were screened using this minitransposon. Some of them were developmental deficient. Eight strains were selected for Southern Blot analysis and the randomness of the transposition was confirmed. The stableness of the transposons was also confirmed by relaxed cultivation for several generations. The transposon system constructed here has provided a novel approach for the function genomic analysis of Streptomyces.更多还原