维吉尼亚链霉菌IBL14中sviscsn基因簇功能分析

发布时间：2018-06-04 00:06

本文选题：维吉尼亚链霉菌IBL14 + I-B-svi型CRISPR-Cas系统　；参考：《安徽大学》2017年硕士论文

【摘要】：细菌和古细菌中广泛存在着成簇的、规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)被称之为 CRISPR 系统,这个CRISPR作为原核生物中可获得性免疫功能器官而存在。外源的遗传物质(病毒DNA或质粒)片段经剪切加入到CRISPR的repeat间隔中成为spacer,当外源遗传物质再次侵入细菌时,CRISPR通过特异性识别定向攻击并降解其外源遗传物质,从而达到免疫的作用。维吉尼亚链霉菌IBL14(Streptomyces virginiae IBL14)是实验室分离得到的一株能以多种甾体化合物为唯一碳源进行生长的菌株,是迄今为止所发现的唯一能生物催化薯蓣皂苷元(diosgenin)F-环(C-25羟基化)的微生物。实验室对该菌株进行了全基因测序和功能分析预测,发现染色体存在18个CRISPR位点,其中两个CRISPR位点之间存在一个Cas蛋白群,顺序为cas7,cas5,cas3,cas4,cas 1,cas2,将其命名为I-B-svi型CRISPR-Cas系统,已证实可对自身染色体进行基因编辑。同时发现该菌株染色体中的4个结构基因:细胞色素p450基因(svu001)、RmLC-like基因(svicup02)、细胞色素p450基因(svu015)及一氧化氮合成酶基因(svinos01),相邻连接成一个转录子,命名为sviscsn基因簇。该论文旨在研究维吉尼亚链霉菌IBL14中细胞色素P450的结构与功能,特别是对sviscsn基因簇中4个基因通过自身的I-B-svi型CRISPR-Cas系统对其基因进行敲除,得到相应的突变株,结合生物转化,生理形态和生物信息学等方法对其酶基因功能进行分析,结合大肠杆菌为宿主构建相应的重组菌株,通过重组菌对精氨酸为底物进行生物转化,产物分离与鉴定,确定其转化途径中酶基因功能。本课题取得的研究成果如下:1)生物信息学软件分析发现菌株IBL14基因组中存在一个sviscsn基因簇;2)经I-B-svi型CRISPR-Cas系统自敲除获得五株突变菌株 LBL14Δsvu001、IBL14Δsvicup02、IBL14Δsvu015,IBL14Δsvinos01,IBL14Δsviscsn,发现该五株突变菌株在培养基中生长速度;3)有趣的是:sviscsn基因簇顺序基因敲除导致菌落颜色变化加深,全部敲除的,颜色恢复;4)该4个基因分别克隆于大肠杆菌后得到重组子,经诱导表达和加入精氨酸转化,发现CYP酶Svu001,Svu015可以在L-精氨酸的N原子上引入具有区域选择性和立体专一性的ω-羟基。
[Abstract]:A cluster of regularly spaced short palindrome repeats known as the CRISPR system is present in both bacteria and archaea. This CRISPR exists as an organ of acquired immune function in prokaryotes. The foreign genetic material (virus DNA or plasmid) was cut into the repeat interval of CRISPR to become a spacer. When the exogenous genetic material invaded the bacteria again, CRISPR specifically identified the targeted attack and degraded the exogenous genetic material. To achieve the role of immunity. Streptomyces Virginia (IBL14(Streptomyces virginiae IBL14), a strain isolated from the laboratory, which can grow with multiple steroids as the sole carbon source, is the only biocatalyst for diosgenin F- ring C-25 hydroxylation of diosgenin. The whole gene sequencing and functional analysis of the strain were carried out in the laboratory. It was found that there were 18 CRISPR loci on the chromosome, among which there was a Cas protein group between the two CRISPR loci. The sequence was cas7, cas5, cas3, cas1, cas2, and named I-B-svi CRISPR-Cas system. It has been proved that gene editing of its own chromosome can be carried out. At the same time, four structural genes were found in the chromosomes of the strain: cytochrome p450, rmLC-like and cytochrome p450, and nitric oxide synthase gene, named as sviscsn gene cluster. The purpose of this study was to study the structure and function of cytochrome P450 in IBL14 of Streptomyces Virginia, especially to knockout the four genes in the sviscsn gene cluster by their own I-B-svi CRISPR-Cas system, and to obtain the corresponding mutants, which combined with biotransformation. The function of its enzyme gene was analyzed by physiological morphology and bioinformatics. The corresponding recombinant strain was constructed with Escherichia coli as host. Arginine was biotransformed by recombinant strain, and the product was isolated and identified. The function of enzyme gene in its transformation pathway was determined. The research results obtained in this paper are as follows: 1) Bioinformatics software analysis shows that there is a sviscsn gene cluster 2 in the IBL14 genome of the strain. Five mutant strains LBL14 螖 svu001IBL14 螖 svicup02IBL14 螖 svu015 IBL14 螖 svinos01IBL14 螖 sviscsnwere obtained by self-knockout of I-B-svi CRISPR-Cas system. What's interesting is that knockout of the sequence genes of the 1: svisCSN gene cluster leads to a deeper color change in the colony. All knockout, color recovery 4) the four genes were cloned into Escherichia coli respectively to obtain the recombinant gene, which was induced to express and add arginine transformation. It was found that CYP enzyme Svu001n Svu015 could introduce 蠅 -hydroxyl groups with regioselectivity and stereoselectivity on N-atom of L-arginine.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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