问号钩体56609株基因组特征及钩体转化系统建立

发布时间：2018-03-13 02:34

本文选题：问号钩体　切入点：基因组　出处：《上海交通大学》2014年博士论文　论文类型：学位论文

【摘要】：钩体病是由致病性钩体引起的世界范围内流行的人畜共患病。由于缺乏有效的遗传操作手段,对钩体及钩体病的研究进展一直比较缓慢。已有报道表明问号钩体基因组只含有大小两个染色体(CI和CII),没有其它染色体外遗传物质存在。用高通量测序方法对56609株进行了全基因组测序,发现56609株基因组中除染色体外,还可能含有3个质粒(lcp1、lcp2和lcp3)。为了验证这3个质粒为独立于染色之外的复制子,本研究结合生物信息学分析预测、原位脉冲场凝胶电泳(PFGE)分离、Southern杂交等实验方法,成功证实染色体外质粒的存在,同时还验证了质粒的稳定性,并通过PCR策略排除了其整合进基因组的可能性。同时,应用相似的实验方法,鉴定并纠正了56601株基因组中基因岛LaiGI序列为染色体外质粒Laicp,大小为64kb。基于56609株中三个质粒的复制起始域(rep及parAB基因)序列,在双曲钩体穿梭载体(L.biflex-E.coli)pGKBLe24的基础上通过替换复制起始域而构建了三类不同的问号钩体(L.interrogans-E.coli)穿梭载体,并运用此载体成功转化了双曲Patoc I株、问号钩体56606株和56610株,首次成功构建了问号钩体的复制型载体,为其遗传操作研究奠定了基础。对同种致病性问号钩体56609株、56601株及Fiocruz L1-130株的核酸序列进行比较时发现染色体上存在可能由可移动元件引起的基因组重排。56609株基因组比其它两株大,除了含有3个特异的质粒外,染色体基因组中还存在大片段重复序列。对这三株菌的O抗原基因簇分析表明,由于56609株属于流感伤寒群而56601株及Fiocruz L1-130株均属于黄疸出血群,O抗原区基因簇序列有较大差异,在相似性很高的O抗原区基因簇序列中间存在一段菌株特异性基因序列,可以作为区分不同血清群的靶基因。用丝裂霉素C对56609株进行培养诱导并通过透射电镜观察发现,56609株能被诱导出噬菌体颗粒,通过RT-PCR及Real-Time PCR方法及生物信息学分析结果推测56609株中前噬菌体遗传物质可能为质粒lcp3。同时对问号钩体56606株进行诱导,也发现能够诱导出噬菌体颗粒,说明前噬菌体可能在钩体基因组广泛分布。这也是首次在问号钩体中诱导发现完整的噬菌体颗粒。
[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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：S852.6;R514.4

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