【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后出現的由副溶血性弧菌引起的食物中毒爆發(fā),可能是由‘大流行菌群'引起。同本學者在2003年完成了副溶血性弧菌大流行菌株RIMD2210633的全基因組序列的測定。本研究目的是通過基因芯片技術,針對大量具有代表性的全球性副溶血性弧菌菌株進行基于芯片的比較基因組學研究,以深入認識副溶血性弧菌基因組多態(tài)性和種群的系統(tǒng)發(fā)育。 方法利用副溶血性弧菌全基因組序列,挑選出4770條基因,PCR擴增各基因并將PCR產物純化,點樣制備芯片。設計了兩個質控雜交組合,采用雙色熒光雜交策略,對芯片質量進行評價,并用PCR方法驗證部分芯片結果。針對174株副溶血性弧菌,進行基于芯片的比較基因組學研究。用原核表達系統(tǒng)構建副溶血性弧菌幾種重要毒力相關基因的表達載體。 結果成功的研制了一批副溶血性弧菌全基因組DNA芯片。經質量評價,發(fā)現芯片雜交與理論預期結果以及PCR驗證結果完全一致,證明此芯片質量良好,可以用于后續(xù)的比較基因組雜交。建立了基于DNA芯片的副溶血性弧菌比較基因組學技術平臺,建立了一套系統(tǒng)的芯片數據分析的標準方法。成功構建了六種重要毒力相關基因的原核表達載體,為以后深入研究副溶血性弧菌致病機制打下物質基礎。 我們對174株菌的芯片數據進行了系統(tǒng)進化分析后,得到了174個菌株的種系結構圖——最小生成樹。174株菌被分為了5個群(C1至C5),每個群內的個體在種系發(fā)生和遺傳學上彼此密切相關。C3和C4代表高毒的臨床克隆株,而C5群和無毒的環(huán)境分離株高度相關。C2和C3分別組成了兩個不同的克隆群——‘舊03:K6克隆群'和‘大流行菌群'。c3包括了所有經PCR驗證為大流行菌株的39個菌株(trh~-,tdh~+和GS-PCR~+),C2則包括了12株1996前的舊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)是由舊O3:K6克隆進化而來,進化過程包括toxRS/新序列的出現和基因組島的逐步獲得。研究還發(fā)現了介于大流行菌群和舊O3:K6克隆群之間的一個種系發(fā)育的‘中間型O3:K6進化枝'(trh~-,tdh~- and GS-PCR~+)。174株菌的差異組成的基因占全基因組總數的22%。 結論通過大量菌株的基于芯片的比較基因組學研究,獲得了這些菌株的基因組成概況、種群結構和大流行菌群的進化史。
[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.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R346;R155.3

【引證文獻】

相關碩士學位論文 前2條

1 劉陽;副溶血弧菌和溶藻弧菌快速檢測體系的建立與應用[D];福建農林大學;2012年

2 侯立杰;重慶地區(qū)水產品中副溶血性弧菌的污染情況調查和分離株的毒力因素及藥物敏感性研究[D];西南大學;2013年

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本文編號：2335563