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研究生:李永華
研究生(外文):Yung-Hua Lee
論文名稱:耐冷菌Pseudomonassp.P90產生胞外蛋白質分解脢之調控基因的鑑定
論文名稱(外文):Identification of gene controlling 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:2001
畢業學年度:89
語文別:中文
中文關鍵詞:蛋白質分解脢耐冷菌調控基因
外文關鍵詞:Pseudomonastwo-component system
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最近的一些研究報告指出,Pseudomonas屬細菌胞外酵素活性(如胞外蛋白質分解脢、脂質分解脢)大都由lemA/gacA基因所組成之“two-component regulatory system”所調控。
本實驗室由塔塔加山地分離得到的耐冷菌Pseudomonas sp. P90,具有胞外蛋白質分解脢(protease)及脂質分解脢(lipase)活性。經由轉座子pUT-Tn5(pfm)/Tc誘變後,挑選出一株失去protease及lipase活性之菌株,命名為P90A2。本研究由北方墨點法(Northern blot)實驗得知其protease基因並未轉錄,顯示突變株之喪失胞外的protease與lipase活性並非無法分泌所造成。進一步分析轉座子所攜帶的Tetracycline抗藥基因在P90A2染色體上的插入位置,發現在涵蓋插入點的一段長2691 bp的核甘酸序列上有三個open reading frame(ORF)。選殖此一DNA片段,利用Maxi-cell進行基因表現,發現有一個大小約為50 kDa的蛋白質產生。此結果與從其中一個ORF hypo450預測蛋白質大小相同。針對此hypo450所做的分析工作,發現與許多菌種的未知蛋白(hypothetical protein)相似;針對此ORF的氨基酸的序列加以分析,得知在氨基酸的功能上與two-component regulatory system所屬的蛋白質有類似的區域。但是hypo450具有類似tyrosine kinase的功能,並非類似lemA是屬於histidine protein kinase。因此hypo450是屬於HPR或 RR類似的功能蛋白,或是另外獨立的調控蛋白需要進一步的探討。

Recent resrarch reports that several exo-enzyme activity of genus Pseudomonas were regulated by two-component regulatory system composed of lemA/gacA gene.
Pseudomonas sp. P90 which is a psychotrophic bacterium isolated from Ta-Ta-Ja Taiwan in Mountain had both exocellular protease and lipase activity. After mutagenizing with mini transposon pUT-Tn5/(pfm)Tc a strain P90A2 without exocellular protease and lipase activity were isolated. According to the result of Northern blot, the loss of protease activity was due to no transcription of protease gene but not prohibition of secretion. When analyzed the insertion site of the tetracycline resistance gene of transposon in P90A2 chromosomal DNA, We could find three open reading frames (ORF) in a nucleotide sequence of 2691-bp which spanned to the insertion site. When cloned this DNA fragment and analyzed it’s possible gene expression in maxi-cell, one protein with a molecular weight of about 50 kDa was found. This size was in close agreement with the value calculated for e deduce polypeptide product of ORF hypo450, one of the three open reading frame. The alignment analysis of amino acid sequences showed that the deduced polypeptide product of ORF hypo450 was similar to a few hypothetical proteins of several bacteria. Although it had no similarity with LemA and GacA in amino acid sequence, it had many amino acid sites related to tyrosine kinase function, instead a histidine protein kinase like LemA. Therefore, whether the gene product of ORF hypo450 is belonging to HPK or RR of two component regulatory system, or another independent regulatory protein, need further confirmation.

中文摘要……………………………………… ……………………..1
英文摘要…………………………………………….………………..2
前言…………………………………….……………………………..3
材料方法……………………….……………………………………..7
Ⅰ實驗材料……………….……………………………………………..7
1. 菌種與質體………..……………………………………………..7
2. 藥品……..………………………………………………………..9
3. 培養基配方..……………………………………………………..9
4. 緩衝溶液配方………………………………..…………………..9
Ⅱ實驗方法…………………………………….……………………….13
一、DNA之製備……………………………………………………….14
1.小量質體DNA之抽取…………………………………………….14
2.大量質體DNA之抽取…………………………………………….14
3.染色體DNA之抽取……………………………………………….15
二、質體之構築 (cloning)……………………………………………..15
1.DNA回收…………………………………………………………..15
2.DNA的補齊(fill in) ……………………………………………….16
3.DNA的連接(ligation) ………………………………….………….16
三、細胞的轉型作用 (transformation) ……………..…..….…………16
1.製作勝任細胞 (competent cell) …………………….….…………16
2.轉型作用 (transformation) ……………………………..…………17
3.電孔法(Electroporation) ………………………………..………….17
4.快速質體篩選法……………………………………….…………..17
四、南方墨點雜配法 (Southern blot ) ……………………...….…….18
1.探針(probe)之製備……………………………………....….……..18
2.南方墨點法…………………………………………….…………..18
五、細菌內RNA之純化…………………………………...….………19
六、北方墨點雜配法(northern blot hybridization) ……….…..………20
七、反轉錄聚合脢鏈鎖反應(RT-PCR) ………………….……..…….20
八、以Maxi-cell進行基因表現………………………….….…..…….21
九、蛋白質大量表現與萃取……………………………….…………..22
十、大量表現蛋白質純化步驟…………………………….…....……..22
十一、SDS-PAGE之蛋白質析…..…………………………..…..……22
十三、in situ protease assay ……………………. ………….….………23
結果…………………………………………………………….……….24
一、薰蒸試驗……………………. ……………………...……….24
二、南方墨點法分析……………………. ……………...……….24
三、北方墨點法分析……………………. ……………….……...24
四、跳躍子pUT-Tn5(pfm)/Tc的插入點之DNA定序…………25
五、插入點DNA序列分析……………………. ……….……….25
六、插入點鄰近之序列分析……………………. ………………26
七、反轉錄聚合脢鏈鎖反應……………………. ………………27
八、以Maxi-cell進行基因表現…………………………… ……27
九、ORF hypo450氨基酸序列之嫌水區域分析……………..…28
十、ORF hypo450氨基酸序列之功能性分析………………..…28
討論…………………………………………………………………….29
參考文獻……………………………………………………………….35
圖表…………………………………………………………………….40
附錄…………………………………………………………………….64

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