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dc.description.abstract生物添加法(Bioaugmentaion)與生物刺激法( Biositimulation )是常使用在工程上的生物復育策略方法,然而對於土壤中菌相的特性以及菌種可以分解污染物的能力的資訊不是非常了解。利用傳統的微生物技術可以知道裡面有哪些菌群卻不能知道他們對於復育的能力,本實驗研究突破傳統微生物檢測法之的限制,將偵測的目標鎖定在油污分解相關的開環酵素catechol 1,2-dioxygenase 基因上。設計出可放大本土性油污分解菌種之catechol dioxygenase基因之PCR引子,配合油污土壤DNA萃取技術與real-time PCR定量技術,偵測土壤中所含catechol 1,2-dioxygenase油污分解功能性基因的量以評估現地復育的能力。 Rhodococcus erythropolis是一種可以分解油污的放線菌,Rhodococcus eythropolis CC-BC11原篩選自中興大學土環所楊秋忠實驗室,經實驗測試可以使用Benzene、Toluene、Naphthalene and Catechol等環狀化合物作為唯一碳源,很適合用於污染土壤的生物復育,本研究設計一組針對Rhodococcus erythropolis 開環酵素catechol 1,2- dioxygenase的專一性引子,並使用即時定量PCR定量技術,其偵測極限可以偵測到 103–104 to 108–109 gene copies/reaction,透過catA基因數量與catechol降解的速率關係,可以作為生物復育降解的指標,線性關係係數R2可達0.95以上 ,透過此發展的DGGE與Real-time PCR技術運用在污染土壤實場監測 。zh_TW
dc.description.abstractBioaugmentation and biositimulation are two of the most popular strategies for bioremediation. However, it is difficult to evaluate which kind of bio-treatment is better for the case. Traditional microbial method can show what is inside the soil but not tell what they can do. By detection and quantification of catechol 1,2-dioxygenase genes in oil polluted soil, our method break through the limits by traditional microbial method and tell us the actual information of microbial degradation ability. Based on the catechol 1,2-dioxygenase genes primers designed from Taiwanese local oil degradation strain. This study combines the real-time PCR technique and oil-polluted soil DNA extraction technique to monitor catechol 1,2-dioxygenase copies number. The study can reveal the microbial activities after the bio-treatment is started and can improve the efficiency of bioremediation. Gram-positive Rhodococcus erythropolis, an actinomycete, is kind of oil degrading bacteria. R. erythropolis CC-BC11 was originally isolated from crude oil contaminats soil in C. C. Young labtory. R. erythropolis CC-BC11 can use Benzene, Toluene, Naphthalene and catechol for sole carbon source. R. erythropolis CC-BC11 has the potential as a bioremediation organism. In this study, we designed the specific primer sets for catechol 1,2-dioxygenase of Rhodococcus and one primer set, catABC11-F/R, for R. erythropolis. The new designed primer sets allow the detection of corresponding genotypes in soil with a detection limitation from 103-104 to 108-109 gene copies/reaction. The relationship of catechol degrading rate and catA gene copies number is a good index of bioremediation potential in contaminants site. The relationship coefficient, R2, is up to 0.95. We hope this developed system of real-time PCR technique can apply into field-scale contaminant soilen_US
dc.description.tableofcontents致謝 i 中文摘要 ii Abstract iii 第一章 前言 1 第二章 文獻回顧 4 一、生物添加法(Bioaugmentation) 4 (一)加入已馴化的混菌 4 (二)添加純菌 4 (三)功能性菌種添加基因水平傳播 5 (四)生物添加之後續追蹤 5 二、芳香烴化合物的生物降解 5 (一)Polycyclic aromatic hydrocarbons (PAHs)生物降解 6 (二)Benzene、Toluene、Ethylbenzene、Xylenes (BTEX)生物降解 6 三、芳香烴開環酵素catechol dioxygenase 6 四、石油分解菌種Rhodococcus erythropolis 7 (ㄧ)菌種特徵 7 (二)生物降解與耐受性能力 7 (三)界面活性劑之生成 8 (四)Rhodococcus之其他功能 8 五、Real-time QPCR的運用 8 (一)Real-time QPCR的原理 8 (二)Real-time QPCR的運用 9 六、功能性引子的設計 9 第三章 研究材料及方法 11 一、研究所用菌種篩選及其保存 11 (一)研究目標菌種 11 二、培養基 12 (一)Luria-Bertani(LB)培養基 12 (二)Minimal medium(MM)培養基 12 三、菌種環狀化合物耐受性測試 13 (一)Rhodococcsus sp.環狀化合物耐受性測試 13 (二)芳香烴化合物分解菌的環狀化合物測試 13 四、DNA的製備 13 (一)Genomic DNA的少量製備 13 (二)質體DNA的少量製備 14 (三)土壤中微生物DNA的製備 14 五、核酸洋菜膠體電泳 (agarose gel electrophoresis) 的分析與紀錄 15 六、引子的設計與測試分析 16 (一)引子設計方向 16 (二)功能性基因搜尋與基因親緣關係分析 16 (三)CatA-F/catA-R菌種特異性引子設計 16 (四)C12O-F/C12O-R之菌種廣效性引子設計 17 (五)Rhodococcus erythropolis專一性引子設計 17 (六)Primer專一性測試 17 七、聚合酶鏈反應 (Polymerase chain reaction, PCR) 17 (一)操作方法 17 (二)反應條件 18 八、PCR產物片段回收與純化 18 九、核酸的黏合反應(ligation) 18 十、基因選殖(cloning)與大腸菌轉形 (transformation) 19 (一)製備勝任細胞 19 (二)轉型(trasformation) 19 十一、即時聚合酵素鏈鎖反應(real-time PCR) 20 (一)及時聚合酵素鏈鎖反應條件及操作方法 20 (二) 反應偵測標準曲線(standard curve)絕對定量 20 (三)功能性基因數計算公式 21 十二、Real-time PCR基因數與CFU關係比較 21 (一)液態培養純菌比較 21 (二)菌液於土壤中之比較 22 十三、 HPLC分析 22 十四、變性梯度電泳分析(Denaturing gradient gel electrophoresis,DGGE) 22 (一)DGGE KH100實場土壤菌相分析 23 第四章 研究結果 24 一、芳香烴化合物分解菌的環狀化合物測試 24 二、Rhodococcsus sp.環狀化合物耐受性測試結果 24 三、生物資訊搜尋功能性基因引子的設計與探討 25 (一)CatA-F/catA-R引子對Rhodococcus菌屬適用性測試 25 (二)C12OF2/C12OR菌種廣效性引子之Real-time PCR適用性 26 (三)Rhdococcus erythropolis CC--BC11 CatA基因解序 26 (四)Rhodococcus erythropolis專一性引子catABC11-F/R適用性測試 26 (五)Rhodococcus erythropolis catechol 1,2-dioxygenase操縱組探討 26 四、及時聚合酵素鏈鎖反應(real-time PCR)偵測應用 27 (一)定量標準曲線standard curve 27 (二)Real-time PCR靈敏性測試 27 五、即時聚合酵素鏈鎖反應(real-time PCR)偵測應用 27 (一)純菌CFU與Real-time PCR的定量結果 27 (二)土壤CFU與Real-time PCR的定量結果 28 六、功能性基因數量與環狀化合物降解關係 28 (一)各不同數量菌數與環狀化合物降解情況 28 (二)CatA基因數、平板菌落數CFU、OD值與catechol降解關係 28 七、實場污染土壤樣品分析 29 (一)DGGE分析 29 (二)利用real-time PCR 分析污染土壤樣品 30 第五章 討論與建議 31 一、菌種特異性引子適用性 31 二、Real-time PCR定量結果探討 31 (一)引子間自相黏合之誤差 31 (二)土壤DNA抽取與採樣 32 (三)土壤中PCR抑制物 32 (四)土壤DNA片段大小 32 (五)土壤背景的影響 33 (六)建議 33 第六章 參考文獻 34 圖表附錄 39 表目錄 39 表 2-1、實驗室研究用菌株 39 表 2-2、 CatA功能性基因引子設計菌種表 40 表 2-3、PCR反應溫度條件 41 表 2-4、Real-time PCR反應藥品含量 42 表 2-5、DGGE 膠體配製 43 表 3-1、芳香烴化合物分解菌的環狀化合物測試 44 表 3-2、Rhodococcsus sp.環狀化合物耐受性測試結果 45 表 3-3、本實驗設計之引子 46 表 3-4、純菌CFU與Real-time PCR的功能性基因定量結果 47 表 3-5、土壤CFU與Real-time PCR的功能性基因定量結果 48 表 3-6、 Real-time PCR KH100功能性基因分析結果 49 表 3-7、各時期 TPH C10~C28 降解速率表 50 圖目錄 51 圖 1-1、基因檢測流程( Paul et al., 2005) 51 圖 1-2、各類PAHs好氧性降解途徑(Hamme et al.,2003) 52 圖 1-3、好氧性細菌代謝苯環類化合物途徑(Harayama et al., 1992) 53 圖 1-4、芳香族化合物的好氧性降解途徑 ( Mesarch et al., 2003) 54 圖 1-5、兩種參與芳香烴環狀化合物分解之雙加氧酵素 55 圖 1-6、Aromatic ring-cleavge dioxygenase的分類(Horsman et al., 2005)。 56 圖 1-7、Real-time PCR定量偵測系統 57 圖 2-1、Catechol 1,2-dioxygenase親源演化樹(DANMAN vision 58 圖 2-2、CatA-F/CatA-R引子設計之胺基酸保留區域(Vector NTI 9.0 AligX) 59 圖 2-3、C12O-F/C12O-R之菌種廣效性引子 60 圖 2-4、Rhodococcus erythropolis專一性引子CatABC11-F/ CatABC11-R設計(Vector NTI 9.0 AligX) 61 圖 3-1、菌種Catechol測試比較 62 圖 3-2、Rhodococcus erythropolis含catechol平板培養基測試 63 圖 3-3、CatA-F/catA-R Rhodococci菌屬特異性引子適用性 64 圖 3-4、C12OF2/C12OR菌種廣效性引子之Real-time PCR適用性 65 圖 3-5、Rhdococcus erythropolis CC-BC11序列與比對圖 66 圖 3-6、CatABC11-F/CatABC11-R引子專一適用性測試 67 圖 3-7、Rhodococcus erythropolis catechol 1,2-dioxygenase操縱組 68 圖 3-8、Rhodococcus erythropolis BC11 cat operon 測試 69 圖 3-9 、Rhodococcus erythropolis CC-BC11與R. erythropolis CCM2595比對圖(DANMAN vision 70 圖 3-10、Real-time PCR定量標準曲線standard curve 71 圖 3-11、Real-time PCR靈敏性測試 72 圖 3-12、環狀化合物降解圖 73 圖 3-13、CatA基因數、平板菌落數CFU與OD值與catechol降解關係 74 圖 3-14、污染場址KH100生物復育流程圖(來源成功大學環工系鄭幸雄老師實驗室) 75 圖 3-15、KH100土壤DGGE分析測試 76 圖 3-16、利用Real-time PCR 分析KH100污染土壤樣品。 77 圖 4-1、七株油污分解菌中16S rDNA序列之親緣關係圖(DANMAN vision 78 圖 4-2、Real-time PCR的Melting curve。 79zh_TW
dc.subjectcatechol dioxygenaseen_US
dc.subjectreal-time PCRen_US
dc.titleUsing the functional gene of catechol dioxygenase as a bioremediation marker for crude oil contaminated site.en_US
dc.typeThesis and Dissertationzh_TW
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