本文選題：維吉尼亞鏈霉菌IBL14 + I-B-svi型CRISPR-Cas系統(tǒng)��； 參考：《安徽大學》2017年碩士論文

【摘要】：細菌和古細菌中廣泛存在著成簇的、規(guī)律間隔的短回文重復序列(clustered regularly interspaced short palindromic repeats,CRISPR)被稱之為 CRISPR 系統(tǒng),這個CRISPR作為原核生物中可獲得性免疫功能器官而存在。外源的遺傳物質(病毒DNA或質粒)片段經(jīng)剪切加入到CRISPR的repeat間隔中成為spacer,當外源遺傳物質再次侵入細菌時,CRISPR通過特異性識別定向攻擊并降解其外源遺傳物質,從而達到免疫的作用。維吉尼亞鏈霉菌IBL14(Streptomyces virginiae IBL14)是實驗室分離得到的一株能以多種甾體化合物為唯一碳源進行生長的菌株,是迄今為止所發(fā)現(xiàn)的唯一能生物催化薯蕷皂苷元(diosgenin)F-環(huán)(C-25羥基化)的微生物。實驗室對該菌株進行了全基因測序和功能分析預測,發(fā)現(xiàn)染色體存在18個CRISPR位點,其中兩個CRISPR位點之間存在一個Cas蛋白群,順序為cas7,cas5,cas3,cas4,cas 1,cas2,將其命名為I-B-svi型CRISPR-Cas系統(tǒng),已證實可對自身染色體進行基因編輯。同時發(fā)現(xiàn)該菌株染色體中的4個結構基因:細胞色素p450基因(svu001)、RmLC-like基因(svicup02)、細胞色素p450基因(svu015)及一氧化氮合成酶基因(svinos01),相鄰連接成一個轉錄子,命名為sviscsn基因簇。該論文旨在研究維吉尼亞鏈霉菌IBL14中細胞色素P450的結構與功能,特別是對sviscsn基因簇中4個基因通過自身的I-B-svi型CRISPR-Cas系統(tǒng)對其基因進行敲除,得到相應的突變株,結合生物轉化,生理形態(tài)和生物信息學等方法對其酶基因功能進行分析,結合大腸桿菌為宿主構建相應的重組菌株,通過重組菌對精氨酸為底物進行生物轉化,產(chǎn)物分離與鑒定,確定其轉化途徑中酶基因功能。本課題取得的研究成果如下:1)生物信息學軟件分析發(fā)現(xiàn)菌株IBL14基因組中存在一個sviscsn基因簇;2)經(jīng)I-B-svi型CRISPR-Cas系統(tǒng)自敲除獲得五株突變菌株 LBL14Δsvu001、IBL14Δsvicup02、IBL14Δsvu015,IBL14Δsvinos01,IBL14Δsviscsn,發(fā)現(xiàn)該五株突變菌株在培養(yǎng)基中生長速度;3)有趣的是:sviscsn基因簇順序基因敲除導致菌落顏色變化加深,全部敲除的,顏色恢復;4)該4個基因分別克隆于大腸桿菌后得到重組子,經(jīng)誘導表達和加入精氨酸轉化,發(fā)現(xiàn)CYP酶Svu001,Svu015可以在L-精氨酸的N原子上引入具有區(qū)域選擇性和立體專一性的ω-羥基。
[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.
【學位授予單位】：安徽大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78

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