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研究生:黃兆君
研究生(外文):Jau-Jiun Hwang
論文名稱:酚類資化酵母菌Candidatropicalis宿主載體系統之開發
論文名稱(外文):Development of host-vector system for the phenol-utilizing yeast Candida tropicalis
指導教授:張敏政張敏政引用關係
指導教授(外文):Ming-Jeng Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1993
畢業學年度:81
語文別:中文
論文頁數:85
中文關鍵詞:酚類資化酵母菌酚類資化酵母菌形質轉換營養缺失互補
外文關鍵詞:phenol-utilizing yeastphenol-utilizingyeasttransformation
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Candida tropicalis 是一株具有酚類資化(Phenol-utilizing)能力的不
完全酵母菌,此菌不僅能夠在17 mM(=1500 ppm)高濃度的酚類碳源培養基
裡生長, 更可利用烷類或脂肪酸誘發Peroxisome和b-oxidation pathway
酵素大量增加. 因此無論就探討酚類代謝機制,或是增生之調節作用, 開
發其宿主載體系統實有其必要性.有關宿主載體系統之構築, 我們利用各
種致變劑(mutagen)使 Candida tropicalis 發生突變, 並加以抗生素濃
縮後, 再以 5-Fluoroorotate 具專一性的篩選 orotidine
monophosphate decar- boxylase(ura3)缺失的突變株.我們得到兩株尿密
啶營養需求變異株分別命名為U-6與U-20,其中U-6具有低反突變率的優
點, 故相當適合作為宿主. 至於載體部份則利用 YEp- 13所構築
Candida tropicalis的基因庫形質轉換 (transformation)至
Saccharomyces cerevisiaeSHY-3 (ura3-)中, 選殖一段約5Kb長,可以互
補Saccharomyces cerevisiae之ura3-之 C. tropicalis DNA片段. 將此
質體(pCU-1)形質轉換至 Candidatropicalis U-6 host中而得到ura3+轉
形子(transformant), 其形質轉換率為1~10transformants/ug DNA 將此
轉形子及U-6宿主DNA抽出後進行南方吸漬法(Southern blot)目前證據僅
說明了U-6之ura3相較於野生型而言其Pst I site 已消失至於pCU-1 (內
含ura3)在U-6中是以何種型態存在及複製?此點需進行更進一步實驗方可
得知。我們進一步選殖一段自動複製序列(ARS:Autonomous Replication
Sequence), 可使載體得以在U-6宿主之中自動複製,以及提高其轉形率
進荋ㄗ悝韝隢K的基因選殖利器。 因此我們再以帶有ura3之pUC-19作為載
體,並連接上Candida tropicalis DNA片段所構築的基因庫, 形質轉換至
U-6並嚐試抽取超螺旋鏈型的質體,藉此選殖自動複製序列(ARS),我們從
U-6轉型子抽取出10個質體,但此種質體卻不再具有ARS活性,故此ARS基
因選殖工作目前仍處於一未完成之階段。
Candida tropicalis is an asporogenous phenol-utilizing
yeast. It is able to metabolize 17 m M phenol and acts a
model organism for studies on peroxisome biogenesis. The
development of the host-vector system may be valuable in
elucidating the mechanism of phenol metabolism and the
induction of peroxisome biogenesis for Candida tropicalis The
ura3- mutants were mutagenized and isolated by anystatin
enrichment and 5-Fluoro- orotic acid resistance selection.
Two racil requiring mutants were obtained and designated as
U-6, U-20.Because U-6 reverted to uracil prototrophy at a
frequency of less than 10-7.It appeared to be a suitable
host for transformation. To clone the ura3 gene, we
transformed ura3 deficient S. cerevisiae SHY-3 with the C.
tropicalis YEp-basedgenomic library to the ura3 deficient S.
cerevisiae SHY-3. We obtained a C. tropicalis DNA segment,
about 5 kb in length, that could complement the S.cerevisiae
ura3-deficient host. Plasmid vectors containing this segment
transformed the C. tropicalis mutant host at a frequency of
1-10 transforments per mg of plasmid DNA. With Southern blot
analysis, the data suggested that the ura3 of U-6 lacked the
Pst Isite in comparison with ura3 of wild type C. tropicalis.
To confirm whether the ura3 containing plasmid would integrate
to the host chromosome by homologous recombination, further
experiment is required.In addition to integrative vector, we
would further clone the ARS (Autonomous Replication Sequence)
which confer the replicatingplasmids to replicate autonomously
in the U-6 host cell and raise the yeast transformation
efficiency. In order to clone ARS element, we transformed the
C. tropicalis pUC-ura3 vector-based genomic library to U-6. We
isolated 10 free type plasmids from U-6 transformants, but
these plasmids didn't have the ARS activity . The ARS cloning
remains to be finished.

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