Kigamicin生物合成基因簇中orf60的研究

发布时间：2018-01-19 18:16

本文关键词： kigamicin 生物合成路径 PCR-Targeting　出处：《福建师范大学》2016年硕士论文　论文类型：学位论文

【摘要】：Kigamicin是从放线菌中分离到的具有抗细菌和抗肿瘤活性的天然产物。其独特的生物学活性表现为能够抑制营养饥饿状态的肿瘤细胞,而对正常营养生长状态的细胞没有影响。因此,kigamicin可以作为开发特异性抗癌药物的重要来源。本论文通过对kigamicin的生物合成途径的研究,而研究过程中着重阐明kigamicin合成过程中新物质的化学结构。从而进一步了解kigamicin的化学结构与功能上的对应关系。为了研究orf60在kigamicin的生物合成中的功能,本论文开展了以下工作：首先,我们对orf0基因进行生物信息学分析,推测其编码的蛋白可能是一种单加氧酶。然后我们以PIJ778为模板采用PCR技术扩增出一段与orf60两端同源且带有壮观霉素抗性的置换片段。随后将这段置换片段以及整合型质粒pLL59一起转化到E.coil BW25113/pIJ 790中,通过PCR-Targeting技术我们得到重组质粒并命名为pLL200,通过抗性筛选以及PCR验证,证明orf0基因敲除成功。然后,我们将重组质粒pLL200以及质粒pLL68(含有激活因子)转入天蓝色链霉菌ML154,通过孢子接合转移方法得到了突变菌株ML20068。通过对比突变株M120068和野生株ML154发酵产物HPLC-MS的检测结果,发现突变株ML20068产生了[M+H]/Z=538 和 [M+H]/Z=513的两个新组分。它们与kigamicin具有相同UV吸收光谱。它们可能是Kigamicin生物合成过程中的中间产物或衍生物。我们通过对发酵产物分离纯化得到538组分12.6 mg和513组分9.0 mg。高分辨质谱拟合它们的元素组成,确定538组分的分子式为C 28H27NO10,513组分的分子式为(26H24O11最后通过核磁共振解析其化学结构,我们得到了538组分的化学结构。从而推测orf60在kigamicin的生物合成中的功能。
[Abstract]:Kigamicin is a natural product with anti-bacterial and anti-tumor activity isolated from actinomycetes. Its unique biological activity is that it can inhibit nutritional starvation of tumor cells. But there is no effect on the normal vegetative growth state of the cells. Kigamicin can be used as an important source for the development of specific anticancer drugs. In this paper, the biosynthesis pathway of kigamicin was studied. In order to study the chemical structure and function of kigamicin, the chemical structure of the new substance in the process of synthesis of kigamicin was elucidated in order to understand the corresponding relationship between the chemical structure and the function of kigamicin. Function of f60 in biosynthesis of kigamicin. The main work of this thesis is as follows: firstly, we do bioinformatics analysis of orf0 gene. We speculated that the protein encoded by it might be a monooxygenase. Then we used PIJ778 as template and PCR technique to amplify a displacement fragment with spectinomycin resistance and homologous to both ends of orf60. The replacement fragment and the integrated plasmid pLL59 were transformed into E. coil. In BW25113/pIJ 790. The recombinant plasmid pLL200was obtained by PCR-Targeting technique. It was proved that orf0 gene knockout was successful by resistance screening and PCR validation. We transferred recombinant plasmid pLL200 and plasmid pLL68 (containing activator) into Streptomyces ML154. The mutant strain ML20068was obtained by spore conjugation transfer method. The detection results of the mutant M120068 and the wild strain ML154 fermentation product HPLC-MS were compared. It was found that the mutant ML20068 produced. [M H] / R ZN 538 and. [M. Two new components of H] / R ZN 513. They have the same UV absorption spectrum as kigamicin. They may be intermediate products or derivatives of Kigamicin biosynthesis. We obtained 538 component 12.6 by separating and purifying the fermentation product. Mg and 513 components 9.0 mg / g. Their elemental compositions were fitted by high resolution mass spectrometry. The molecular formula of the 538 component was determined to be C28H27NO10O513. The molecular formula of the 538 component was determined as C28H27NO10O513. Finally, the chemical structure of the 538 component was elucidated by nuclear magnetic resonance (NMR). The chemical structure of the 538 component was obtained and the function of orf60 in the biosynthesis of kigamicin was inferred.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TQ460.1;TQ929

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