本文選題：問號(hào)鉤體　切入點(diǎn)：基因組　出處：《上海交通大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：鉤體病是由致病性鉤體引起的世界范圍內(nèi)流行的人畜共患病。由于缺乏有效的遺傳操作手段,對(duì)鉤體及鉤體病的研究進(jìn)展一直比較緩慢。已有報(bào)道表明問號(hào)鉤體基因組只含有大小兩個(gè)染色體(CI和CII),沒有其它染色體外遺傳物質(zhì)存在。用高通量測(cè)序方法對(duì)56609株進(jìn)行了全基因組測(cè)序,發(fā)現(xiàn)56609株基因組中除染色體外,還可能含有3個(gè)質(zhì)粒(lcp1、lcp2和lcp3)。為了驗(yàn)證這3個(gè)質(zhì)粒為獨(dú)立于染色之外的復(fù)制子,本研究結(jié)合生物信息學(xué)分析預(yù)測(cè)、原位脈沖場凝膠電泳(PFGE)分離、Southern雜交等實(shí)驗(yàn)方法,成功證實(shí)染色體外質(zhì)粒的存在,同時(shí)還驗(yàn)證了質(zhì)粒的穩(wěn)定性,并通過PCR策略排除了其整合進(jìn)基因組的可能性。同時(shí),應(yīng)用相似的實(shí)驗(yàn)方法,鑒定并糾正了56601株基因組中基因島LaiGI序列為染色體外質(zhì)粒Laicp,大小為64kb。基于56609株中三個(gè)質(zhì)粒的復(fù)制起始域(rep及parAB基因)序列,在雙曲鉤體穿梭載體(L.biflex-E.coli)pGKBLe24的基礎(chǔ)上通過替換復(fù)制起始域而構(gòu)建了三類不同的問號(hào)鉤體(L.interrogans-E.coli)穿梭載體,并運(yùn)用此載體成功轉(zhuǎn)化了雙曲Patoc I株、問號(hào)鉤體56606株和56610株,首次成功構(gòu)建了問號(hào)鉤體的復(fù)制型載體,為其遺傳操作研究奠定了基礎(chǔ)。對(duì)同種致病性問號(hào)鉤體56609株、56601株及Fiocruz L1-130株的核酸序列進(jìn)行比較時(shí)發(fā)現(xiàn)染色體上存在可能由可移動(dòng)元件引起的基因組重排。56609株基因組比其它兩株大,除了含有3個(gè)特異的質(zhì)粒外,染色體基因組中還存在大片段重復(fù)序列。對(duì)這三株菌的O抗原基因簇分析表明,由于56609株屬于流感傷寒群而56601株及Fiocruz L1-130株均屬于黃疸出血群,O抗原區(qū)基因簇序列有較大差異,在相似性很高的O抗原區(qū)基因簇序列中間存在一段菌株特異性基因序列,可以作為區(qū)分不同血清群的靶基因。用絲裂霉素C對(duì)56609株進(jìn)行培養(yǎng)誘導(dǎo)并通過透射電鏡觀察發(fā)現(xiàn),56609株能被誘導(dǎo)出噬菌體顆粒,通過RT-PCR及Real-Time PCR方法及生物信息學(xué)分析結(jié)果推測(cè)56609株中前噬菌體遺傳物質(zhì)可能為質(zhì)粒lcp3。同時(shí)對(duì)問號(hào)鉤體56606株進(jìn)行誘導(dǎo),也發(fā)現(xiàn)能夠誘導(dǎo)出噬菌體顆粒,說明前噬菌體可能在鉤體基因組廣泛分布。這也是首次在問號(hào)鉤體中誘導(dǎo)發(fā)現(xiàn)完整的噬菌體顆粒。
[Abstract]:Leptospirosis is a worldwide epidemic of zoonosis caused by pathogenic leptospirosis. The research progress on leptospirosis and leptospirosis has been slow. It has been reported that the genome of Leptospira contains only two chromosomes in size, CI and CII.There is no other genetic material outside the chromosome. 56609 was sequenced by high-throughput sequencing. The whole genome was sequenced. Three plasmids, lcp1, lcp2 and lcp3, were found in the genome of 56609 strains. In order to verify that the three plasmids are replicas independent of staining, we combined bioinformatics analysis to predict the three plasmids. In situ pulsed field gel electrophoresis (PFGE) isolation and Southern blotting were used to successfully confirm the existence of plasmids outside chromosomes and the stability of plasmids, and the possibility of integration into genomes was excluded by PCR. Using a similar experimental method, the gene island LaiGI sequences of 56601 strains were identified and corrected as outer chromosomal plasmids (64 kb). Based on the replication initiation domain of three plasmids in 56609 strains, the sequence of parAB gene was identified and corrected. Three different types of Leptospira Leptospira L.interrogans-E.coli shuttle vectors were constructed on the basis of the shuttle vector L.biflex-E.coliparum pGKBLe24. By replacing the replication initiation domain, the shuttle vectors of L.interrogans-E.coli were successfully transformed into hyperbolic Patoc I strains, 56606 and 56610 Leptospira strains. The replicative vector of Leptospira question mark was successfully constructed for the first time. By comparing the nucleic acid sequences of 56609 Leptospira homogenicus strains and Fiocruz L1-130 strains, we found that the genome rearrangement. 56609 gene may be caused by movable elements on chromosome. The group is larger than the other two. In addition to containing three specific plasmids, there are also large repeats in the genome of the chromosomes. Because 56609 strains belong to influenza typhoid group and 56601 strains and Fiocruz L1-130 belong to jaundice haemorrhage group, there is great difference in the sequence of gene cluster of O antigen region, and there is a specific gene sequence in the sequence of O antigen region gene cluster with high similarity. 56609 strains were cultured and induced by mitomycin C and the phage particles were induced by transmission electron microscopy. The results of RT-PCR and Real-Time PCR and bioinformatics analysis suggested that the genetic material of prephage in 56609 strains might be plasmid lcp3. at the same time, 56606 strains of Leptospira question mark could be induced, and the phage particles could be induced. It is also the first time to induce the discovery of complete phage particles in Leptospira interrogans, indicating that the prophage may be widely distributed in the genome of Leptospira.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：S852.6;R514.4

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本文編號(hào)：1604448