創傷弧菌廣泛分佈於�h暖海域，可以在環境中自由營生，或對人及魚類造成感染。我們利用 MLST 技術建立在台灣分離的創傷弧菌菌株之系統發生樹，發現大部份由台灣養殖吳郭魚所分離的菌株極為相近，形成以 93U204 為代表的群集。 93U204 群集與人類臨床菌株 YJ016 位在樹上同樣的群集，而環境分離株 K02 與吳郭魚分離株 86573B，以及環境分離株 Kau 71 與鱸魚分離株 93U262，則分別位在較遠的進化枝上。我們利用 SSCP 技術分析環境菌株與吳郭魚分離株在轉錄相關基因、胺基酸代謝或運送相關基因、無機離子代謝相關基因、訊息傳遞相關基因及表面膜蛋白 / 毒力因子等 5 大類基因上的多樣性，發現在訊息傳遞相關基因及表面膜蛋白 / 毒力因子 2 大類基因有較高的基因多樣性。我們挑選每大類多樣性高的基因共 12 種，進行 PAML 軟體分析發現其中 10種基因不受到天擇作用，顯示挑選的 5 大類基因在創傷弧菌族群中的遺傳多樣性主要由於於中性選汰所造成的。而 ompR 基因是受到正向選汰，且發現部份創傷弧菌環境或魚病分離株擁有相同的胺基酸序列，顯示正向選汰作用在這兩種分離株能同時存在的海水環境，藉由 ompR 基因功能推測物體的附著能力、生物膜形成能力及移動能力，在創傷弧菌適應海水環境有較大的重要性

Vibrio vulnificus is ubiquitous in warm marine environment. This bacterial species can be either free-living or infect human and fish hosts as a pathogen. We reconstruct the phylogenetic relationship of V. vulnificus strains isolated in Taiwan based on MLST analysis, and showed that most of the tilapia isolates form a cluster represented by strain 93U204. The 93U204 cluster has high relatedness to human clinical isolate YJ016 in the MLST tree. The cluster formed by environmental isolate K02 and tilapia isolates 86573B, and the cluster formed by environmental isolate Kau 71 and perch isolate 93U262, are more distantly related to the 93U204 cluster. Using SSCP, we analyzed the genetic diversity in five groups of genes among environmental and tilapia isolates, including genes involved in transcription, amino acid metabolism and transport, inorganic ion metabolism, signal transduction, membrane proteins and virulence factors. Most tilapia strains share the same allele in potB gene alleles, but 6 alleles were found in environmental isolates, indicating potB may be selected in tilapia isolates. Tilapia strain 86573B and perch isolate 93U262 are more closely related to the 93U204 cluster in trpD gene tree than in the MLST tree, suggesting that fish-pathogenic strains are more similar in their trpD gene. Analysis of trpD genes with PAML program has shown that trpD gene was under positive selection. Among the five groups of genes analyzed, evidence of selection was only found in amino acid metabolism and transport genes. This suggests that adaptation in these genes may be important in the evolution of tilapia-pathogenic V. vulnificus.

摘要................................................................I
Abstract............................................................II

第一章 研究背景 ....................................................1
1.1 創傷弧菌 (Vibrio vulnificus)....................................1
1.2 微生物分子分型 (typing) 相關研究................................2
1.3 創傷弧菌分型相關研究............................................3
1.4 天擇的研究方法..................................................6
1.5 單鏈構像多態性技術 (Single strand conformation polymorphism，簡稱SSCP) ....................................................................7
1.6 研究目的........................................................8

第二章 創傷弧菌環境菌株的分離與MLST系統發生樹的建構.................9
2.1 緒論............................................................9
2.2 材料與方法......................................................10
2.3 結果............................................................14
2.4 討論............................................................15

第三章 SSCP分析創傷弧菌分離株不同基因...............................16
3.1 緒論............................................................16
3.2 材料與方法......................................................18
3.3 結果............................................................19
3.4 討論............................................................22

第四章 基因樹的建構與 MLST 系統發生樹的比較.........................24
4.1 緒論............................................................24
4.2 材料與方法......................................................25
4.3 結果............................................................27
4.4 討論............................................................32

參考文獻............................................................61

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