摘要
格蘭氏陰性菌Pseudomonas屬胞外酵素和次級代謝物的產生通常是藉由GacS與GacA所組成的〝two-component regulatory system〞來調控。本實驗室從台灣中部塔塔加山區土樣中分離得到一株能產生胞外蛋白質分解酶及脂質分解酶的耐冷菌---Pseudomonas sp. P90也具有gacS和gacA基因。近來已有研究發現，Sigma factor σS (RpoS)與RNA結合蛋白(RsmA)也會參與調控胞外酵素和次級代謝產物的產生。本論文實驗根據RpoS及RsmA的胺基酸序列中保守性最高的區域設計引子，進行聚合酶連鎖反應來檢測P90，將所得到的DNA片段定序確定為rpoS及rsmA基因後，再將片段接入載體上，利用基因重組交換的方式得到兩株rpoS與rsmA基因破壞的突變株P90rsmA-及P90rpoS-，據此再利用自體黏接選殖到rpoS及rsmA的基因全長。酵素活性的定量分析實驗發現rpoS與rsmA兩個基因的突變會增加P90胞外蛋白質分解酶活性的表現，若使RsmA在野生株P90和突變株P90rsmA-細胞中大量表現，則會降低胞外蛋白質分解酶的活性。因而確定rpoS及rsmA基因也參與調控P90胞外蛋白質分解酶活性的表現。另外，本實驗室早先曾將P90以跳躍子pUT-Tn5(pfm)/Tc誘變，分離得到一株胞外蛋白質分解酶與脂質分解酶活性同時缺失的突變株-P90A2，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苷酸序列上有一段長1350 bp，可能轉譯450個胺基酸的open reading frame，將其命名為ORF450，而Tcr插入點則介於序列上第282 nt與第283 nt之間。前人進行RT-PCR反應與西方墨點法實驗均不能證明有基因的RNA或蛋白質產物產生。若利用同質重組的方式（homologous recombination）將突變株P90A2染色體DNA上被Tcr插入的ORF450置換回原本的ORF450序列，則可以使突變株恢復表現胞外蛋白質分解酶與脂質分解酶的活性。因此，本實驗研究也嘗試改變ORF450的核苷酸序列以定出此序列的功能性範圍。實驗後發現分別在ORF450序列上第914 nt~第915 nt與第1167 nt~第1168 nt之間做插入破壞得到的兩株突變株P90pOK596與P90A68，仍然具有胞外蛋白質分解酶與脂質分解酶的活性，而在第661 nt~第662 nt之間被插壞的突變株P90pOK344，則完全失去胞外蛋白質分解酶與脂質分解酶的活性。因此而將ORF450功能性位置縮短到第914 nt之前的位置。從實驗結果推測除了GacS、GacA、RpoS與RsmA之外，P90尚有ORF450第914 nt之前的DNA序列參與調控整個胞外蛋白質分解酶與脂質分解酶活性的表現。

目錄
中文摘要……………………………………………………………………1
英文摘要……………………………………………………………………3
前言…………………………………………………………………………5
材料方法……………………………………………………………………11
Ⅰ實驗材料…………………………………………………………………11
一.菌種與質體……………………………………………………………11
二.藥品藥品、酵素及放射線同位素……………………………………11
三.培養基…………………………………………………………………11
四.試劑與緩衝溶液………………………………………………………11
Ⅱ實驗方法………………………………………………………………16
一、DNA之製備……………………………………………………………16
1.小量質體DNA之抽取……………………………………………………16
2.大量質體DNA之抽取……………………………………………………16
3.染色體DNA之抽取………………………………………………………17
二、質體之構築 (cloning)……………………………………………17
1.限制酶的切割分析……………………………………………………17
2.DNA回收…………………………………………………………………18
3.DNA的補齊(fill in) …………………………………………………18
4.DNA的連接(ligation) ………………………………………………18
三、細胞的轉型作用 (transformation) ………………………………19
1.製作勝任細胞 (competent cell) …………………………………19
2.轉型作用 (transformation) ………………………………………19
3.電孔法(Electroporation) …………………………………………19
4.快速質體篩選法………………………………………………………20
四、聚合酶連鎖反應………………………………………………………20
五、洋菜膠體電泳分析……………………………………………………21
六、南方墨點雜配法(Southern blot)…………………………………21
1.探針(probe)之製備……………………………………………………21
2.南方墨點法……………………………………………………………21
七、細菌RNA之純化………………………………………………………22
八、北方墨點雜配法(northern blot hybridization)………………22
九、酵素活性分析…………………………………………………………23
1.蛋白質含量測定………………………………………………………23
2. 酵素活性測定…………………………………………………………23
3. SDS-PAGE之蛋白質析…………………………………………………24
十、in situ protease assay …………………………………………24
結果…………………………………………………………………………26
一、Pseudomonas sp. P90 rpoS的基因選殖、分析與突變……………26
(一) Pseudomonas sp. P90的rpoS的基因選殖………………………26
(二) Pseudomonas sp. P90的rpoS的基因序列分析…………………27
(三) Pseudomonas sp. P90 rpoS突變株的篩選………………………28
二、Pseudomonas sp. P90 rsmA的基因選殖、分析與突變……………29
(一) Pseudomonas sp. P90的rsmA的基因選殖………………………29
(二) Pseudomonas sp. P90的rsmA的基因序列分析…………………31
(三) Pseudomonas sp. P90 rsmA突變株的篩選………………………32
三、 RsmA在Pseudomonas sp. P90大量表現的結果……………………33
四、突變株P90rsmA-之互補測試…………………………………………33
五、Pseudomonas sp. P90染色體DNA上ORF450位置與胞外蛋白質分解酶活性表現之關係……………………………………………………………34
(一)ORF450核苷酸序列是否存在於其它Pseudomonas屬細菌的染色體DNA上？………………………………………………………………………34
(二) P90菌株染色體DNA上ORF450 RNA之偵測…………………………35
(三) P90菌株染色體DNA上ORF450序列分析……………………………35
六、胞外蛋白質分解酶的酵素活性測定…………………………………37
(一)突變株：P90rpoS —和P90rsmA-以及轉型株：P90(pBBR1MCS2)、P90 (pBBR1MCS2-rsmA+) 和P90rsmA- (pBBR1MCS3-rsmA+)等之酵素活性分析…………………………………………………………………………37
(二) ORF450基因改變突變株P90A2、P90A68、P90pOK344以及P90pOK596之酵素活性分析…………………………………………………39
討論…………………………………………………………………………40
參考文獻……………………………………………………………………48
圖表…………………………………………………………………………54
附錄…………………………………………………………………………111

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