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標題: Gordonia terrae CC-NAPH129-6菌株對萘分解特性研究及定量偵測方法之建立
Characterization of naphthalene degradation and quantitative detection of Gordonia terrae strain CC-NAPH129-6
作者: 林俊良
Lin, Chun-Liang
關鍵字: Polycyclic aromatic hydrocarbons
出版社: 生命科學系所
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摘要: 多苯環芳香族化合物 (polycyclic aromatic hydrocarbons, PAHs) 廣泛存於環境中,由於其具有致癌性與致突變性對人體的健康造成重大的威脅,因此針對污染的環境進行整治復育已成為現階段最重要的工作。本研究從柴油污染的土壤分離出具分解萘 (naphthalene) 能力的菌株Gordonia terrae CC-NAPH129-6。將Gordonia terrae CC-NAPH129-6菌株接種 7.8×107 CFU/ml 及 6.7×105 CFU/ml 的初始菌數於含 30 mg/l 萘之最小無機鹽液態培養基,分別經過 18 及 24 小時,能將其降解至偵測極限 (0.1mg/l) 以下。利用針對革蘭性陽性菌所設計之引子 RnarAa124 /RnarAa1275 進行聚合酶鏈鎖反應,能成功增殖到 1054 bp 核苷酸片段,經序列比對與 Rhodococcus sp. P400之naphthalene dioxygenase large subunit (narAa) 有具有最高的相似度 99.6%。從Pseudomonas屬與Rhodococcus屬之相似於naphthalene dioxygenase large subunit 基因種系樹狀圖顯示, G. terrae CC-NAPH129-6 菌株的 naphthalene dioxygenase α subunit 的基因與 Rhodococcus 屬的 naphthalene 群叢最為接近。此結果顯示naphthalene dioxygenase α subunit 的基因可能由 Rhodococcus 屬經水平傳播至 G. terrae CC-NAPH129-6 菌株。以 gyrB 基因設計G. terrae CC-NAPH129-6 菌種特異性引子 GTSF/GTSR,利用 G. terrae CC-NAPH129-6 親緣關係接近的 Gordonia 屬的11株菌株確定引子特異性。在土壤及地下水中 G. terrae CC-NAPH129-6 菌株之 DNA濃度或菌數量與CT值具有良好的線性關係,其偵測極限分別為 32 ρg及24 ρg 的 DNA 濃度與 5 × 104 CFU/ml 及 7.6 × 104 CFU/g soil 的菌數量。本研究成果將可提供快速及準確的監測方法以利於應用 G. terrae CC-NAPH129-6 菌株於污染場址中,進行生物復育時菌數的變動的監測。
Polycyclic aromatic hydrocarbons (PAHs), widely distributed environmental toxics, are potential carcinogenic and mutagenic chemicals to human health. Since PAHs tend to accumulate at soil, bioremediation through soil microorganisms is of choice. The Gordonia terrae strain CC-NAPH129-6 was isolated form diesel oil-contaminated soil for the capability to degrade naphthalene. While 7.8×107 CFU/ml or 6.7×105 CFU/ml culture in naphthalene-containing medium (30 mg/l) with strain CC-NAPH129-6 could degrade naphthalene completely with detection limit at 0.1 mg/l in 18 hours or 24 hours, respectively. Using the PCR primer RnarAa124 /RnarAa1275, designed from Rhodococcus naphthalene dioxygenase large subunit gene (narAa) region, the DNA fragment with 1054 bp was amplified with chromosomal DNA of CC-NAPH129-6 as template. The narAa of stain CC-NAPH129-6 shares 99.6% similarity with narAa of Rhodococcus sp. P400. And the phylogenetic tree of the naphthalene dioxygenase α subunit-like genes indicated that the narAa of strain CC-NAPH129-6 was clustered with Rhodococcus sp. It indicated the possibility of horizontal gene transfer of naphthalene dioxygenase from Rhodococcus to Gordonia terrae strain CC-NAPH129-6 which encountered the insult of naphthalene. Specific primers were designed for the gyrB gene region, and the specificity was confirmed with 11 related Gordonia genus. The linear relationship between the threshold fluorescence (CT) value and the quantity of G. terrae strain CC-NAPH129-6 genomic DNA or cell density was measured. The detection limit for CC-NAPH129-6 real-time PCR assay was 32 ρg DNA and 5 × 104 CFU/ml for groundwater samples or 24 ρg of DNA and 7.6 × 104 CFU/g for soil samples. Investigation shown here could be applied to monitor the in situ bioremediation of G. terrae CC-NAPH129-6.
其他識別: U0005-2308200616130600
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