阿霉素生物合成基因簇的克隆和苜蓿中华根瘤菌基因敲除载体的构建

发布时间：2018-07-07 23:35

本文选题：阿霉素 + 生物合成基因簇　；参考：《南京师范大学》2017年硕士论文

【摘要】：本研究工作分为两个部分,第一部分为阿霉素生物合成基因簇大片段克隆。阿霉素是从一种从链霉菌Streptomyces peucetius ATCC 29050菌株中提取的具有一定抗肿瘤作用的蒽环类抗生素,由于现存的化学合成法易造成浪费和污染。所以根据文献报道,本课题尝试设计实验通过异源表达生产阿霉素来取代传统的化学合成方式。本研究首先将S. peucetius ATCC 29050阿霉素生物合成基因簇的各个基因片段通过PCR技术扩增,获得的目的片段通过不同的限制酶处理后,连接至不同的载体。形成带有单基因的克隆,用限制性内切酶处理获得的单基因克隆。回收片段后,通过片段连接方式和其它的单基因克隆相连,将两段基因拼接在一起,用相应的抗生素进行筛选,形成一个新克隆。依次叠加,逐步拼接成为一个目的基因大片段,完成整个基因簇的克隆,最终的目标是实现异源表达。实验将基因簇分成两段,分两部分同时进行,设计用58个克隆完成基因簇拼接,已完成55个,从pDS01至pDS56,从起始到结束,大部分基因已克隆至载体。一旦所有基因被克隆并被拼接到载体中,就可以尝试异源表达来获得阿霉素。本研究工作的第二个部分为苜蓿中华根瘤菌基因敲除载体的构建,并用已构建完成的重组质粒尝试进行基因敲除。对苜蓿中华根瘤菌(Sinorhizobium meliloti)菌株Rm1021的全基因组已测序,本研究尝试用重组工程手段对其四个关键基因进行敲除。与其他基因敲除方法相比,重组工程能使用短片段同源臂高效率地催化体内同源重组反应,能精确、快速的完成传统基因敲除技术无法做到的复杂基因操作。实验构建了多个含有redαβ重组酶基因的质粒。并尝试用含lacIq基因取代lacI基因,LacIq较LacI能更有效控制目的基因表达。从pLS3304至pLS3308,不仅用lacIq基因取代lacI基因,并且增加了 ter片段,重组的严谨性也越来越高。具体方法是首先制备含有能够表达相关重组酶基因质粒的S. meliloti Rm1021电转感受态细胞,将通过OE-PCR获得的重组片段转入感受态细胞中,均匀涂布于含卡那霉素的抗性平板进行筛选,然后再通过PCR验证是否得到卡那霉素抗性基因的重组目的菌株。但是,在尝试了多种方法后,仍未获得突变型敏感菌株,进一步实验还在进行中。尽管实验未能达到敲除基因的目的,但是探索了S. meliloti Rm1021的初步重组工程条件,为以后的实验打下了基础。
[Abstract]:This research is divided into two parts. The first part is the large fragment cloning of adriamycin biosynthesis gene cluster. Adriamycin is a kind of anthracycline antibiotic which is extracted from Streptomyces peucetius ATCC 29050 strain and has certain anti-tumor effect. It is easy to waste and pollute because of the existing chemical synthesis method. Therefore, according to literature reports, this paper attempts to design experiments to produce adriamycin by heterologous expression instead of traditional chemical synthesis. In this study, each gene fragment of S. peucetius ATCC 29050 adriamycin biosynthesis gene cluster was amplified by PCR technique, and the obtained target fragment was ligated to different vectors after different restriction enzyme treatment. A single gene clone was obtained by restriction endonuclease treatment. After the fragments were recovered, the two genes were spliced together and screened with the corresponding antibiotics to form a new clone by linking the fragments to other single gene clones. The whole gene cluster was cloned and the final goal was to achieve heterologous expression. The gene cluster was divided into two segments and divided into two parts. The gene cluster was spliced with 58 clones, 55 of which had been cloned from pDS01 to pDS56, and most of the genes were cloned to the vector from the beginning to the end. Once all genes are cloned and spliced into the vector, heterologous expression can be attempted to obtain adriamycin. The second part of this study was to construct the gene knockout vector of Rhizobium sativa and try to knockout with the constructed recombinant plasmid. The whole genome of Sinorhizobium meliloti strain Rm1021 was sequenced. Compared with other gene knockout methods, recombination engineering can efficiently catalyze homologous recombination reaction in vivo using short fragment homologous arm, and can perform complex gene operation accurately and quickly. Several plasmids containing red 伪尾 recombinant enzyme gene were constructed. It was also attempted to replace LacIq gene with LacIq gene and to control the target gene expression more effectively than Laci gene. From pLS3304 to pLS3308, not only lacIq gene was substituted for Laci gene, but also ter fragment was increased. The specific method is to first prepare the S. meliloti Rm1021 electroporative cells containing plasmids capable of expressing the relevant recombinant enzyme genes. The recombinant fragments obtained by OE-PCR are transferred into the receptive cells and evenly coated on the kanamycin resistant plate for screening. Then the recombinant target strain of kanamycin resistance gene was obtained by PCR. However, after several methods have been tried, no mutant sensitive strains have been obtained, and further experiments are still under way. Although the experiment failed to achieve the purpose of knockout, the preliminary recombination engineering conditions of S.meliloti Rm1021 were explored, which laid a foundation for further experiments.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

【参考文献】