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研究生:毛傳忠
研究生(外文):Mao, Chuan-Jong
論文名稱:腸炎弧菌擬鐵調節基因之選殖與特性研究
論文名稱(外文):Cloning and characterization of a fur-like gene from the Vibrio parahaemolyticus
指導教授:黃顯宗黃顯宗引用關係
指導教授(外文):Hin-Chung Wong
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1996
畢業學年度:85
語文別:中文
論文頁數:83
中文關鍵詞:腸炎弧菌擬鐵基因編碼核酸序列胺基酸序列
外文關鍵詞:Vibrio parahaemolyticusfur-like geneComplete coding sequenceAmino acid sequence
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由於鐵離子在細胞的呼吸作用、酵素活性及核酸的生成扮演重要的角色,
使得它成為大部分微生物生存所不可或缺的物質;然而在有氧環境中攝取
過量的鐵可能會對細胞造成毒害,必須適當調控其攝取量。負責調節攝取
鐵機制的基因最早在大腸桿菌中發現,稱為 fur基因,隨著研究發展逐漸
發現大腸桿菌中受到 fur基因調控的基因非常多,幾乎所有與攝鐵相關的
基因都包含在內,甚至有些與攝鐵無關的基因也受到調控。已知腸炎弧菌
的致病性會受環境中鐵離子濃度的影響,此影響可能是透過 fur-like 基
因來表現,因此希望在選殖到腸炎弧菌的 fur-like 基因之後,對於腸炎
弧菌的致病性及其他許多生理現象都能做更深入的研究。目前至少在 12
種菌株當中發現有 fur-like 基因的存在,而且必此間具有保守性。本研
究中先比對霍亂弧菌和大腸桿菌 fur基因,選擇高相似度的區域,以或霍
亂弧菌染色體作為模板,用 PCR方式製備成 0.46kb 大小的核酸片段作為
選殖 fur-like 基因的探針。取腸炎弧菌染色體核酸以限制酵素 EcoRI切
割成片段,進行電泳及南式轉漬法後,以製備的探針進行雜合試驗,將所
得的反應區域的片段接到載體 pUC19中,以探針經雜合試驗篩選選殖株後
進行核酸定序,得到一段完整的編碼核酸序列,和大腸桿菌 fur基因編碼
序列比對有 71.1%的相似度,與霍亂弧菌有 80.0%相似度;若將此核酸序
列推演出胺基酸序列,與大腸桿菌 fur基因推演之胺基酸序列相似度為
79.7%,與霍亂弧菌之相似度為 92.6%。
Iron has involed in many essential processes inside cells, e.g.
respiration, enzyme activity, ribonucleotide synthesis, etc.
However, iron is also a potentially toxic element because it can
catalyze the formation of detrimental hydroxyl radicals which
can damage virtually all cell constituents. Therefore,
microorganisms have to regulate the level of intracellular iron.
The gene that controls the iron-uptake system is called fur
(ferric uptake regulation) gene and it was first cloned and
sequenced in Escherichia coli. The fur gene coordinates the
regulation of genes involved in iron uptake. The level of iron
also affected the pathogenicity of V. parahaemolyticus, thus the
fur-like gene may also play certain role in the virulaence of
this pathogen. By comparing E. coli fur gene with V. cholerae
fur-like gene, a 0.46kb conserved fragment was identified.
Polynucleotide probe was generated by polymerase chain reaction
on the region by using V. cholerae template. Southern
hybridization of V. parahaemolyticus chromosomal DNA digested by
EcoRI showed a single band under low low stringency condition.
This band was eluted, cloned into pUC19 and screened by using
the same probe. Nucleotide sequence was performed by Sanger
dideoxy sequencing method, and the sequence was analyzed by PC/
GENE software. Results showed that a complete coding sequence
(CDS) of the fur-like gene was obtained. The amino acid sequence
(AAS) derived from the nucleotide sequence comprised 149 amino
acid residues. Comparing with the E. coli fur gene, the fur-like
gene of V. parahaemolyticus show 71.1 and 79.9% similarity in
terms of CDS and AAS, respectively. Comparing with the Vibrio
cholerae fur-like gene, the fur-like gene of V. parahaemolyticus
show 80.0 and 92.6% similarity in terms of CDS and AAS,
respectively.
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