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研究生:涂純星
研究生(外文):Twu, Chung-Shing
論文名稱:Erwiniachrysanthemi之idg基因啟動子及其受PecS蛋白調節之分析
論文名稱(外文):Promoter activity of Erwinia chrysanthemi idgA gene and its regulation controlled by PecS protein
指導教授:黃秀珍黃秀珍引用關係
指導教授(外文):Huang, Hsiao-Chen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:52
中文關鍵詞:核甘酸果膠
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  Erwinia chrysanthemi (Ech) 為植物病原菌,會分泌多种的胞外植物細胞壁分解酵素引起軟腐病害。某些E. chrysanthemi之菌株在yeast-extract dextrose calcium-carbonate (YDC)培養基上常可產生藍色素而使菌落成為藍色,這藍色結構為5,5'-DIAMINO-4,4'-DIHYDROXY-3,3'-diazadiphenoquione的indigoidine化合物。由Ech RA3B菌株選殖一段與藍色素合成有關的6kb EcoRI片段,此片段含有pecS、pecM、idgA及idgB基因。為了解藍色素基因特性,構築RA3B-idgA、RA3B-idgB、RA3B-pecM非極性突變株,結果顯示這些菌株在YDC培養基中RA3B-idgA為無色、RA3B-idgB為淺藍色、RA3B-pecM為深藍色。利用T7RNA polymerase dependent系統表現idgA,idgB及pecS基因,證實有基因產物。由互補實驗顯示idgA及idgB基因可能屬於同一轉錄單位。由primer extension 分析顯示:idgA基因起始密碼上游第114個核?酸”C”為轉錄起始點。又Band-shift assay 顯示PecS蛋白呆專一性地結合上idgA基因上游區域。以luxAB為報導基因系統可偵測idgA上游區域,確實具有啟動子活性並且在E.coli細胞內證實PecS蛋白可抑制idgA基因啟動子活性。在Ech RA3B菌體內多量表現的pecS基因可抑制細菌藍色素及果膠分解酵素的產生,證實PecS蛋白為idgA基因的負調控因子。


  Erwinia chrysanthemi (Ech) causes bacterial soft rot disease on several crops by secreting extracellular pectinase and cellulase. A spontaneous mutant Ech RA3B produces tremendous amounts of a black-blue pigment (indigoidine, 5,5' -diamino-4,4'-dihydroxy-3,3'-diazadiphenoquinone) on yeast-extract dextrose calciumcarbonate (YDC) medium. A 6kb EcoRI DNA fragment containing pecS, PECm, idgA, and idgB genes involved in blue pigment biosynthesis was cloned from Ech RA3B strain. Three non-polar mutants, RA3B-idgA, RA3B-IDgB, and RA3B-pecM, were constructed by insertion of nptII gene to investigate blue pigmentation. The mutants RA3B-idgA, RA3B-idgB, and RA3B-pecM cultured on YDC medium produced white, pale-blue, and dark-blue colonies, respectively. The idgA, idgB and pecS genes can be overexpressed by T7 RNA polymerase dependent system. Complementation assay, primer extension and band-shift assay yielded several observations. (1) idgA and idgB genes are likely to be organized in the same transcriptional unti (2) A transcription initiation site located at the 114th nucleotide (C) upstream of the idgA gene from the ATG site. (3) PecS protein can bind upstream region of the idgA gene specifically. Furthermore, the luciferase genes, luxA and luxB, were used as reporter genes to monitor the promoter activity The results showed that a promoter activity was detected at the upstreacm region of idgA and the activity of the promoter was inhibited by PecS protein in E. coli. Mutiple copies of pecS gene present in Ech RA3B supressed the production of the bacterial blue pigment and pectinase, indicating that PecC protein is a negative regulator of the idgA gene.

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