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研究生:林惠敏
研究生(外文):Lin Huei-Ming
論文名稱:影響耐冷菌Pseudomonassp.P90胞外蛋白質分解生合成之調控基因的分析與比較
論文名稱(外文):Analysis and Comparison of Gene Regulating Extracellular Protease Production from a Psychrotrophic Bacterium Pseudomonas sp. P90
指導教授:溫福賢
指導教授(外文):Fu-Shyan Wen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:84
中文關鍵詞:耐冷菌調控基因調控系統
外文關鍵詞:psychrotrophic bacteriumregulating genestwo-component regulatory system
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原核生物常在感知環境的刺激後採取反應,其間環境訊息的轉導通常是由〝two-component regulatory system〞中一對蛋白質來調控。此系統包括一個兼具sensor功能與kinase活性的sensory kinase,以及一個response regulator。Pseudomonas屬細菌主要即是由GacS與GacA構成的系統來調控胞外酵素、抗生物質或毒性物質等的產生。先前本實驗室從台灣中部塔塔加山區土樣中分離得到的一株能產生胞外蛋白質分解及脂質分解的耐冷菌---Pseudomonas sp. P90。本實驗根據GacS及GacA胺基酸序列中保守性最高的區域設計引子,然後以聚合連鎖反應來檢測P90,結果顯示P90的染色體DNA亦有gacS和gacA基因。
先前本實驗室曾將P90以跳躍子pUT-Tn5(pfm)/Tc誘變,分離得到一株胞外蛋白質分解與脂質分解活性同時缺失的突變株-P90A2。經分析其染色體DNA上tetracycline抗藥基因插入點旁的DNA片段,發現在涵蓋插入點的核酸序列上有一段長1350 bp,可能轉譯450個胺基酸的open reading frame(ORF),將其命名為ORF hypo450。抽取P90的RNA進行RT-PCR反應,發現ORF hypo450並不會產生基因產物RNA。以pET21a選殖ORF hypo450,純化產生的融合蛋白並製成血清,再以 SDS-PAGE分離P90全細胞蛋白質後進行西方墨點法實驗,結果並無任何免疫反應的訊號產生。另以泛寄主載體選殖染色體DNA上包含ORF hypo450的2619 bp DNA片段後,送回突變株進行互補測試,發現無法將原有胞外蛋白質分解與胞外脂質分解的活性補回。但若利用基因間同質重組置換(homologous recombination)將完整的ORF hypo450置換回突變株P90A2與P90pOK421,則可以恢復其蛋白質分解與脂質分解的活性。因此,推測除了GacS與GacA之外,P90尚需ORF hypo450 DNA序列來參與調控其胞外蛋白質分解與脂質分解活性的表現。

Prokaryotes can respond to a variety of environmental stimuli by means of systems generally composed of two proteins. The first protein is sensory kinase, which has sensor function and kinase activity. The second protein is response regulator. In the genus Pseudomonas, a conserved two-component regulatory system consisting of the GacS and GacA has a decisive role in the control of the production of extracellular enzymes, antibiotics or toxins. Previously, Pseudomonas sp. P90, a gram-negative psychrotrophic bacterium isolated from Ta-Ta-Ja Mountain of middle Taiwan, can produce extracellular proteolytic and lipolytic activity. We designed a pair of primers based on the strickly conserved region of GacS and GacA amino acid sequences, in order to use PCR to identity P90. The results revealed that P90 has gacS and gacA gene.
Previously, following transposon pUT-Tn5(pfm)/Tc mutagenesis, an extracellular protease- and lipase-negative mutant, P90A2, was isolated from P90. When we analyzed the insertion site of the tetracycline resistance gene of transposon in P90A2 chromosomal DNA, we could find an open reading frame (ORF) with 1350 bp, designated ORF hypo450. We analyzed the transcript of ORF hypo450 by RT-PCR, the results suggested that ORF hypo450 has no transcript with the size similar to that of the ORF hypo450 coding region. We cloned the pET21a vector with ORF hypo450, then purified the fusion protein and made into serum. Western blotting analysis of total protein separated by SDS-PAGE demonstrated that there is no hypo450 protein in wild type P90. Further, proteolytic activity was not restored to protease- and lipase-negative mutants harboring broad-host-range vector which containing 2619 bp DNA fragment of ORF hypo450 fragment. But the proteolytic activity and lipolytic activity of the mutants(P90A2 and P90pOK421), was restored by double crossover the ORF hypo450 by homologous recombination. Therefore, we inferred that beside the GacS and GacA, ORF hypo450 also regulated the expression of the extracellular protease and lipase activity of P90.

中文摘要……………………………………… ……………………….1
英文摘要…………………………………………….……………….…2
前言…………………………………….…………………………….…4
材料方法……………………………………………………………..…8
Ⅰ實驗材料……………………………………………………………...8
1. 菌種與質體………………………………………………………..8
2. 藥品、酵素及放射性同位素……………………………………..8
3. 培養基……………………………………………………………..8
4. 試劑與緩衝溶液配方……………………………………………..9
5. 引子…………………………………………………………….…13
Ⅱ實驗方法……………………………………………………………..15
一、DNA之製備………………………………………………………15
1.小量質體DNA之抽取…………………………………………...15
2.大量質體DNA之抽取…………………………………………...15
3.染色體DNA之抽取……………………………………………...16
4.快速質體篩選法…………………………………………………..17
二、質體之構築與選殖 (cloning)……………………………………17
1.限制的切割分析………………………………………………..17
2.DNA回收…………………………………………………………17
3.DNA的補齊(fill in) ………………………………………………18
4.DNA的連接(ligation) ……………………………………………18
三、聚合連鎖反應(polymerase chain reaction, PCR)……….…19
四、洋菜膠體電泳分析(agarose gel electrophoresis)…………….19
五、細胞的轉型作用 (transformation) ……………………………...19
1.勝任細胞 (competent cells)之製備……………………………...19
2.轉形作用 (transformation) ………………………………………20
六、電孔法(electroporation) ………………………………………….20
七、南方轉漬雜配法 (Southern blotting hybridization ) ……………20
1.探針(probe)之製備………………………………………………..20
2.南方墨點法………………………………………………………..21
八、細菌RNA之純化………………………………………………...21
九、北方墨點雜配法(northern blotting hybridization) ………………22
十、反轉錄聚合連鎖反應(RT-PCR) ………………………………23
十一、蛋白質之大量表現…………………………………………….23
十二、蛋白質之SDS-PAGE電泳分析………………………………24
十三、蛋白質回收…………………………………………………….25
十四、抗體製備……………………………………………………….25
十五、西方墨點法…………………………………………………….25
結果……………………………………………………………………….27
一、P90 gacS基因的選殖…….……………………...………………27.
二、P90 gacA基因的選殖與分析……………………………………28
(一) gacA基因之選殖…………………………………………28
(二) GacA胺基酸序列之分析………………………………...28
三、反轉錄聚合連鎖反應(RT-PCR)…………………………..29
四、ORF hypo450蛋白之偵測………………………………………29.
(一) ORF hypo450蛋白抗體的製備…………………………29
(二) 西方墨點法分析…………………………………………32
五、互補測試…………………………………………………………33
六、基因間同質重組置換(homologous recombination)…………34.
七、轉型株之反轉錄聚合連鎖反應(RT-PCR)………………..34
八、以 primer hypo450NdeⅠ和primer hypo450HindⅢ進行聚合連鎖反應………………………………………………………………35
討論………………………………………………………………………..36
參考文獻……………………………………………………………….….44
圖表………………………………………………………………………..50
附錄………………………………………………………………………..83

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