Pectobacterium carotovorum subsp. carotovorum (Pcc)屬於腸道菌科中革蘭氏陰性桿狀菌。此菌株感染植物時會產生果膠酶使植物組織成為濕黏狀態而引起軟腐病。為了與其他親屬相近的菌種競爭會產生細菌素(Bacteriocin)，Pcc會生產低分子量細菌素(LMWB)，透過紫外光的刺激和葡萄糖的濃度會誘導carocin基因表現，但是到目前為止在低分子量細菌素carocin基因調控的部分，尚無相關報導發表。
cyclic-di-GMP是細菌內普遍存在的二級訊號，調控細菌內ㄧ系列的生理功能，像是生物運動性、生物膜形成、細胞分化等，diguanylate cyclases (DGC)會催化兩分子的GTP產生cyclic-di-GMP；而cAMP receptor protein (CRP)在大腸桿菌中通常會與cAMP形成複合體而調控基因，影響了許多啟動子的轉錄作用。在本實驗室先前的報導中指出，crp突變株和dgc突變株的低分子量細菌素carocin基因皆無法表現，因此本研究目的在於探討c-di-GMP與CRP之協同作用對低分子量細菌素carocin基因的調控機制。
在實驗研究中，將dgc與crp基因分別構築於pGEM®-T Easy載體中，命名為pG-DGC和pG-CRP。利用插入抗藥性基因的方式形成了dgc或crp重組基因片段後，利用同質互換的方式將3F3菌株中的dgc或是crp基因阻斷。我們透過RT-PCR及細菌素方法分析3F3野生株與3F3/Δcrp突變株。在細菌素測試中，3F3/Δcrp突變株的抑制半徑小於野生株3F3菌株；而透過RT-PCR的實驗中，在野生株中可以看到caroS2K基因(925-bp)存在，而caroS2K基因在3F3/Δcrp突變株時，仍然正常轉錄，但是caroS3I基因(267-bp)在3F3/Δcrp突變株中，無法正常轉錄。
根據實驗結果我們認為在Pcc菌株中crp基因可能是調控caroS3K細菌素表現，因此我們推斷細菌素Carocin S2和Carocin S3的應為不同基因所調控。

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a phytopathogenic enterobacterium responsible for soft rot disease, which characterized by extensive maceration of the affected plant tissue. Pcc also produces one or more antibacterial substances called bacteriocins, which enhance their competitiveness with other related rival species. Some of the Pcc strains produce low-molecular-weight bacteriocin (LMWB). The LMWB of Pectobacterium species was induced by Ultraviolet exposure and glucose. To date, little is known about the regulation mechanism of bacteriocin production.
Cyclic di-GMP (c-di-GMP) is a second messenger that regulates diverse cellular processes in bacteria, including motility, biofilm formation, cell-cell signaling, and host colonization. c-di-GMP is synthesized by diguanylate cyclases (DGCs) from two molecules of GTP. The cAMP receptor protein (CRP) complex (cAMP-CRP) is a global regulator of gene expression. The CRP of Escherichia coli is a dimer made up of identical subunits. It influences transcription from a number of promoters in Escherichia coli. The previous reports from our laboratory the regulation factors, DGC and CRP, play an important role in expression mechanism of Carocin in Pcc.In this study, we want to known that how is the regulatory mechanism of carocin gene throgh cyclic dimeric guanosine monophosphate (c-di-GMP) and cAMP receptor protein (CRP) cooperation.
First, dgc gene was amplified by PCR and subcloned into pGEM®-T Easy vector, and to from the plasmid pG-DGC. The plasmid pG-CRP was made by the same way. Both of dgc-defective and crp-defective were made by using dephosphorylation of vector DNA method. the dgc or crp mutant was acquired from homologues recombination method. And then, we analyzed the mRNA expression level by RT-PCR experiment and bactericion assay. In bacteriocin assay, the size of inhibition zone around strain 3F3/Δcrp was smaller than that around the wild-type strain 3F3. In mRNA level, the presence of the 925-bp amplicon revealed that caroS2K was being transcribed in the cell. The 3F3/Δcrp strain, which was an ampicillin insertional mutant, still could transcribe caroS2K, but the presence of the 267-bp amplicon revealed that caroS3I was being transcribed in the cell . The 3F3/Δcrp strain could not transcribe caroS3I.
Based on the data presented in this study, we think maybe crp is a novel effecter for caroS3K transcription. It indicate that Carocin S2 and Carocin S3 are different gene to regulate. It will be of interest to study the role of dgc in the carocin gene in the future.

謝誌 i
摘要 ii
Abstract iii
目次 v
表目次 vii
圖目次 viii
名詞縮寫表 ix
第一章 緒論 1
一、細菌性軟腐病(Bacterial soft- rot disease) 1
二、細菌素(Bacteriocin) 2
三、recA修復調控SOS反應機制 4
四、啟動子(Promoter) 5
五、細菌素基因調控機制探討 5
六、研究目的 7
第二章 材料藥品與實驗方法 9
一、材料、藥品 9
(一)、細菌培養液 9
(二)、Genomic DNA之分離 9
(三)、plasmid DNA 之分離 10
(四)、聚合酶鏈反應(Polymerase chain reaction, PCR) 11
(五)、選殖基因與表現載體限制性酵素切割與分析 12
(六)、選殖基因與表現載體之接合作用 13
(七)、勝任細胞(competent cell)製備 14
(八) 、轉型作用(transformation) 15
(九) 、RNA之分離與純化 15
(十) 、甲醛agarose配置與電泳分析 16
(十一) 、反轉錄聚合酶鏈反應( RT-PCR ) 16
(十二) 、電穿孔法 (Electroporation) 16
(十三) 、Pectobacterium carotovorum subsp. carotovorum. 菌株細菌素測試 17
(十四) 、十二烷基硫酸鈉聚丙烯醯胺凝膠電泳分析( SDS-PAGE ) 17
(十五) 、西方墨點法 (Western) 18
(十六) 、Dephosphorylation of vector DNA 19
二、 實驗方法與儀器 20
(一)、Genomic DNA之分離 20
(二)、plasmid DNA之分離 20
(三)、大量質體的分離 21
(四)、聚合酶鏈反應(Polymerase chain reaction, PCR) 22
(五)、選殖基因與表現載體限制性酵素切割與分析 22
(六)、DNA片段之回收與純化 23
(七)、選殖基因與表現載體之接合作用 24
(八)、勝任細胞(competent cell)製備 24
(九)、轉型作用 (transformation) 25
(十)、RNA之分離與純化 25
(十一)、甲醛agarose配置與電泳分析 26
(十二)、反轉錄聚合酶鏈反應( RT-PCR ) 26
(十三)、電穿孔法轉型作用(Electroporation) 26
(十四)、Pectobacterium carotovorum subsp. carotovorum. 菌株細菌素測試 27
(十五)、十二烷基硫酸鈉聚丙烯醯胺凝膠電泳分析(SDS-PAGE) 27
(十六)、西方墨點法(Western) 28
(十七)、Dephosphorylation of vector DNA 30
(十八)、實驗儀器 30
第三章 結果分析 33
一、CRP融合蛋白大量表現確認 33
二、CRP抗體免疫反應測試確認 33
三、CaroS2K抗體免疫反應測試確認 33
四、3F3與H-rif-8-6野生株的Reverse Transcriptase PCR (RT-PCR) assay 34
五、3F3野生株細菌素測試 34
六、3F3/Δcrp突變株的置備 34
七、3F3野生株與3F3/Δcrp突變株細菌素(Bacteriocin)測試 35
八、3F3野生株與3F3/Δcrp突變株的RT-PCR assay 35
第四章 討論 37
附錄一a 42
參考文獻 61

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