副溶血性弧菌全基因组DNA芯片和比较基因组学研究
发布时间：2018-11-16 12:52
【摘要】： 背景副溶血性弧菌是引起全球食物中毒的重要致病菌,1996后出现的由副溶血性弧菌引起的食物中毒爆发,可能是由‘大流行菌群'引起。同本学者在2003年完成了副溶血性弧菌大流行菌株RIMD2210633的全基因组序列的测定。本研究目的是通过基因芯片技术,针对大量具有代表性的全球性副溶血性弧菌菌株进行基于芯片的比较基因组学研究,以深入认识副溶血性弧菌基因组多态性和种群的系统发育。 方法利用副溶血性弧菌全基因组序列,挑选出4770条基因,PCR扩增各基因并将PCR产物纯化,点样制备芯片。设计了两个质控杂交组合,采用双色荧光杂交策略,对芯片质量进行评价,并用PCR方法验证部分芯片结果。针对174株副溶血性弧菌,进行基于芯片的比较基因组学研究。用原核表达系统构建副溶血性弧菌几种重要毒力相关基因的表达载体。 结果成功的研制了一批副溶血性弧菌全基因组DNA芯片。经质量评价,发现芯片杂交与理论预期结果以及PCR验证结果完全一致,证明此芯片质量良好,可以用于后续的比较基因组杂交。建立了基于DNA芯片的副溶血性弧菌比较基因组学技术平台,建立了一套系统的芯片数据分析的标准方法。成功构建了六种重要毒力相关基因的原核表达载体,为以后深入研究副溶血性弧菌致病机制打下物质基础。 我们对174株菌的芯片数据进行了系统进化分析后,得到了174个菌株的种系结构图——最小生成树。174株菌被分为了5个群(C1至C5),每个群内的个体在种系发生和遗传学上彼此密切相关。C3和C4代表高毒的临床克隆株,而C5群和无毒的环境分离株高度相关。C2和C3分别组成了两个不同的克隆群——‘旧03:K6克隆群'和‘大流行菌群'。c3包括了所有经PCR验证为大流行菌株的39个菌株(trh~-,tdh~+和GS-PCR~+),C2则包括了12株1996前的旧03:K6菌株(trh~+,tdh和GS-PCR~-)。大流行菌群f,1996后‘新'03:K6和它的衍生菌株04:K68,O1:K25,01:KUT和06:K18)是由旧O3:K6克隆进化而来,进化过程包括toxRS/新序列的出现和基因组岛的逐步获得。研究还发现了介于大流行菌群和旧O3:K6克隆群之间的一个种系发育的‘中间型O3:K6进化枝'(trh~-,tdh~- and GS-PCR~+)。174株菌的差异组成的基因占全基因组总数的22%。 结论通过大量菌株的基于芯片的比较基因组学研究,获得了这些菌株的基因组成概况、种群结构和大流行菌群的进化史。
[Abstract]:Background Vibrio parahaemolyticus is an important pathogen causing global food poisoning. The outbreak of food poisoning caused by Vibrio parahaemolyticus after 1996 may be caused by 'pandemic bacteria'. The whole genome sequence of Vibrio parahaemolyticus pandemic strain RIMD2210633 was sequenced in 2003. The purpose of this study was to study the microarray based comparative genomics of a large number of representative global Vibrio parahaemolyticus strains by gene chip technology. In order to understand the genome polymorphism and population phylogeny of Vibrio parahaemolyticus. Methods 4770 genes were selected from the whole genome sequence of Vibrio parahaemolyticus, each gene was amplified by PCR, the PCR product was purified, and the microarray was prepared. Two quality control hybrid combinations were designed and the chip quality was evaluated by using two-color fluorescence hybridization strategy. Some of the chip results were verified by PCR method. A microarray based comparative genomics study was carried out on 174 strains of Vibrio parahaemolyticus. Expression vectors of virulence related genes of Vibrio parahaemolyticus were constructed by prokaryotic expression system. Results A batch of whole genome DNA microarrays of Vibrio parahaemolyticus were successfully developed. After quality evaluation, it was found that the chip hybridization was consistent with the expected theoretical results and the PCR verification results. It was proved that the chip was of good quality and could be used for subsequent comparative genomic hybridization. A platform for comparative genomics of Vibrio parahaemolyticus based on DNA chip was established. The prokaryotic expression vectors of six important virulence related genes were successfully constructed, which laid a solid foundation for the further study of pathogenic mechanism of Vibrio parahaemolyticus. Based on the phylogenetic analysis of 174 strains, the phylogenetic diagram of 174 strains was obtained. 174 strains were divided into 5 groups (C1 to C5). Individuals in each group are closely related to phylogeny and genetics. C3 and C4 represent highly virulent clinical clones. C5 and non-toxic environmental isolates were highly correlated. C2 and C3 consisted of two distinct clone groups' old 03:K6 clone 'and' pandemic bacteria'. C3 included all strains identified by PCR as pandemic strains. 39 strains (trh~-,) Tdh~ and GS-PCR~, C2 included 12 old 03:K6 strains (trh~, tdh and GS-PCR~-) before 1996. The new 03:K6 and its derivative strain 04K68O1: K25O01KUT and 06:K18) were derived from the old O3:K6 clones after 1996. The evolution process included the emergence of new toxRS/ sequences and the gradual acquisition of genomic islands. It was also found that a strain of 'intermediate O3:K6 evolutionary branch' (trh~-,tdh~- and GS-PCR~) developed between the pandemic flora and the old O3:K6 clone. 174 strains of bacteria with different composition genes accounted for 22% of the total genome. Conclusion based on the microarray comparative genomics studies of a large number of strains, the gene composition, population structure and evolution history of these strains were obtained.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346;R155.3
本文编号：2335563
[Abstract]:Background Vibrio parahaemolyticus is an important pathogen causing global food poisoning. The outbreak of food poisoning caused by Vibrio parahaemolyticus after 1996 may be caused by 'pandemic bacteria'. The whole genome sequence of Vibrio parahaemolyticus pandemic strain RIMD2210633 was sequenced in 2003. The purpose of this study was to study the microarray based comparative genomics of a large number of representative global Vibrio parahaemolyticus strains by gene chip technology. In order to understand the genome polymorphism and population phylogeny of Vibrio parahaemolyticus. Methods 4770 genes were selected from the whole genome sequence of Vibrio parahaemolyticus, each gene was amplified by PCR, the PCR product was purified, and the microarray was prepared. Two quality control hybrid combinations were designed and the chip quality was evaluated by using two-color fluorescence hybridization strategy. Some of the chip results were verified by PCR method. A microarray based comparative genomics study was carried out on 174 strains of Vibrio parahaemolyticus. Expression vectors of virulence related genes of Vibrio parahaemolyticus were constructed by prokaryotic expression system. Results A batch of whole genome DNA microarrays of Vibrio parahaemolyticus were successfully developed. After quality evaluation, it was found that the chip hybridization was consistent with the expected theoretical results and the PCR verification results. It was proved that the chip was of good quality and could be used for subsequent comparative genomic hybridization. A platform for comparative genomics of Vibrio parahaemolyticus based on DNA chip was established. The prokaryotic expression vectors of six important virulence related genes were successfully constructed, which laid a solid foundation for the further study of pathogenic mechanism of Vibrio parahaemolyticus. Based on the phylogenetic analysis of 174 strains, the phylogenetic diagram of 174 strains was obtained. 174 strains were divided into 5 groups (C1 to C5). Individuals in each group are closely related to phylogeny and genetics. C3 and C4 represent highly virulent clinical clones. C5 and non-toxic environmental isolates were highly correlated. C2 and C3 consisted of two distinct clone groups' old 03:K6 clone 'and' pandemic bacteria'. C3 included all strains identified by PCR as pandemic strains. 39 strains (trh~-,) Tdh~ and GS-PCR~, C2 included 12 old 03:K6 strains (trh~, tdh and GS-PCR~-) before 1996. The new 03:K6 and its derivative strain 04K68O1: K25O01KUT and 06:K18) were derived from the old O3:K6 clones after 1996. The evolution process included the emergence of new toxRS/ sequences and the gradual acquisition of genomic islands. It was also found that a strain of 'intermediate O3:K6 evolutionary branch' (trh~-,tdh~- and GS-PCR~) developed between the pandemic flora and the old O3:K6 clone. 174 strains of bacteria with different composition genes accounted for 22% of the total genome. Conclusion based on the microarray comparative genomics studies of a large number of strains, the gene composition, population structure and evolution history of these strains were obtained.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346;R155.3
【引证文献】
相关硕士学位论文 前2条
1 刘阳;副溶血弧菌和溶藻弧菌快速检测体系的建立与应用[D];福建农林大学;2012年
2 侯立杰;重庆地区水产品中副溶血性弧菌的污染情况调查和分离株的毒力因素及药物敏感性研究[D];西南大学;2013年
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