Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21200
標題: 棒狀桿菌 mFP - 抗性基因的選殖及利用選位突變法分析
Cloning of mFP-Resistant Gene from Corynebacterium glutamicum
作者: 詹明勳
Chan, Ming-Shun
關鍵字: Corynebacterium glutamicum;棒狀桿菌;phenylalanine;site-directed mutagensis;苯丙胺酸;選位突變法
出版社: 分子生物研究所
摘要: 
本研究以 Corynebacterium glutamicum CCRC11384 為親株, 突變篩選
對 mFP 有不同抗性的變異株。分析變異株與親株的 DAHP synthase 活性
,顯示 DAHP synthase 已解除回饋抑制作用。 利用 PCR 從變異株合
成 DAHP synthase 基因,雖可互補 E. coli CCRC50350 之 DAHP
synthase 缺陷性狀,但無法使 C. glutamicum 之轉形株對 mFP 產生抗
性。從 mFP 抗性變異株 C. glutamicum NCU846 的基因庫中選殖到一
mFP 抗性基因經 DNA序列證實此基因片段含有 prephenate dehydratase
基因, pheA'。 利用 PCR 從各突變株中合成 pheA 基因(包含啟
動子區域), 殖入 C. glutamicum 中, 各轉形株皆可對 mFP 產生
抗性, 不過卻不能互補 E. coll CCRC51737 之 prephenate
dehydratase 缺陷性狀。對各 pheA 基因進行 DNA 序列分析, 與原親
株比對變異點。 經由選位突變法發現, 將 Leu-230 轉變為
phenylalanine、 Ala-308 轉變為 arginine 或 Gly-224轉變為
alanine, 並不能改變 phenylalanine 對 prephenate
dehydratase 的回饋抑制作用。 然而, 將 Gly-224 轉變為 histidine
、 tryptophan 或是轉變為 threonine 並配合 Arg-202 轉變為
histidine以及轉變為 alanine 後配合著 Phe-230 與 Arg-308 的轉
變, 則可使 prephenate dehydratase 的活性不再受 phenylalanine 回
饋抑制。 這些結果顯示, Gly-224 在 prephenate dehydratase 回饋抑
制位中,扮演著重要的角色。

m-Flurophenylalanine (mFP)-resistant mutants of Coryne-
bacterium glutamicum was isolated by nitrosoguanidine muta-
genesis.The DAHP synthase genes of mFP-resistant C.
glutamicum was amplified by Polymerase Chain Reaction
technique and trans-formed into mFP-sensitive C.
glutamicum LS1183. The trans-formants could not resist to mFP
although the DAHP synthase activities in those mFP-
resistant C. glutamicum were insensitive to the feedback
inhibition by phenylalanine and tyrosine. Molecular cloning
of mFP-reista nt gene revealed that mutated prephenate
dehydratase, encoded by the pheA gene, conferred the mFP-
resistant phenotype upon C. glutamicum. To determine the
amino acid residues which might involve in the phenylalanine-
mediated feedback inhibition of prephenate dehydratase,
the pheA gene was modified by site-directed mutagenesis.
The changes of Leu-230 to Phe, Ala-308 to Arg-308 and Gly-224
to Ala-224 did not alter the phenylalanine sensitivity of the
prephenate dehydratase. However, Trp-224 or His-224 replace
ment, Thr-224 combined with His-202 substitutions and Ala-224
combined with Phe-230 and Arg-308 replacements rendered
prephenate dehydratase insensitive to phenylalanine. These
results suggested that Gly-224 located at the C-terminal
region of prephenate dehydratase maybe played a key role in
the feedback regulation of prephenate dehydratase by
phenylalanine.
URI: http://hdl.handle.net/11455/21200
Appears in Collections:分子生物學研究所

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