本文選題：阿霉素 + 生物合成基因簇�。� 參考：《南京師范大學(xué)》2017年碩士論文

【摘要】：本研究工作分為兩個(gè)部分,第一部分為阿霉素生物合成基因簇大片段克隆。阿霉素是從一種從鏈霉菌Streptomyces peucetius ATCC 29050菌株中提取的具有一定抗腫瘤作用的蒽環(huán)類抗生素,由于現(xiàn)存的化學(xué)合成法易造成浪費(fèi)和污染。所以根據(jù)文獻(xiàn)報(bào)道,本課題嘗試設(shè)計(jì)實(shí)驗(yàn)通過(guò)異源表達(dá)生產(chǎn)阿霉素來(lái)取代傳統(tǒng)的化學(xué)合成方式。本研究首先將S. peucetius ATCC 29050阿霉素生物合成基因簇的各個(gè)基因片段通過(guò)PCR技術(shù)擴(kuò)增,獲得的目的片段通過(guò)不同的限制酶處理后,連接至不同的載體。形成帶有單基因的克隆,用限制性內(nèi)切酶處理獲得的單基因克隆。回收片段后,通過(guò)片段連接方式和其它的單基因克隆相連,將兩段基因拼接在一起,用相應(yīng)的抗生素進(jìn)行篩選,形成一個(gè)新克隆。依次疊加,逐步拼接成為一個(gè)目的基因大片段,完成整個(gè)基因簇的克隆,最終的目標(biāo)是實(shí)現(xiàn)異源表達(dá)。實(shí)驗(yàn)將基因簇分成兩段,分兩部分同時(shí)進(jìn)行,設(shè)計(jì)用58個(gè)克隆完成基因簇拼接,已完成55個(gè),從pDS01至pDS56,從起始到結(jié)束,大部分基因已克隆至載體。一旦所有基因被克隆并被拼接到載體中,就可以嘗試異源表達(dá)來(lái)獲得阿霉素。本研究工作的第二個(gè)部分為苜蓿中華根瘤菌基因敲除載體的構(gòu)建,并用已構(gòu)建完成的重組質(zhì)粒嘗試進(jìn)行基因敲除。對(duì)苜蓿中華根瘤菌(Sinorhizobium meliloti)菌株Rm1021的全基因組已測(cè)序,本研究嘗試用重組工程手段對(duì)其四個(gè)關(guān)鍵基因進(jìn)行敲除。與其他基因敲除方法相比,重組工程能使用短片段同源臂高效率地催化體內(nèi)同源重組反應(yīng),能精確、快速的完成傳統(tǒng)基因敲除技術(shù)無(wú)法做到的復(fù)雜基因操作。實(shí)驗(yàn)構(gòu)建了多個(gè)含有redαβ重組酶基因的質(zhì)粒。并嘗試用含lacIq基因取代lacI基因,LacIq較LacI能更有效控制目的基因表達(dá)。從pLS3304至pLS3308,不僅用lacIq基因取代lacI基因,并且增加了 ter片段,重組的嚴(yán)謹(jǐn)性也越來(lái)越高。具體方法是首先制備含有能夠表達(dá)相關(guān)重組酶基因質(zhì)粒的S. meliloti Rm1021電轉(zhuǎn)感受態(tài)細(xì)胞,將通過(guò)OE-PCR獲得的重組片段轉(zhuǎn)入感受態(tài)細(xì)胞中,均勻涂布于含卡那霉素的抗性平板進(jìn)行篩選,然后再通過(guò)PCR驗(yàn)證是否得到卡那霉素抗性基因的重組目的菌株。但是,在嘗試了多種方法后,仍未獲得突變型敏感菌株,進(jìn)一步實(shí)驗(yàn)還在進(jìn)行中。盡管實(shí)驗(yàn)未能達(dá)到敲除基因的目的,但是探索了S. meliloti Rm1021的初步重組工程條件,為以后的實(shí)驗(yàn)打下了基礎(chǔ)。
[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 偽 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.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78

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