臺灣博碩士論文加值系統

已知烷烴在環境中穩定並且不易生物降解。 此外，由於它是致癌的並且在體內積累，因此對人類的健康具有危害。 有幾種從環境中除去烷烴的方法，其中，生物修復是一種使用微生物的修復方法，被認為是環保，低成本和具有成本效益的。 在這項研究中，使用從北海道油田的土壤中提取的DNA（其含有高濃度的碳氫化和物和PAH），該微生物群落具有可降解烷烴和PAH的微生物群落，期待將來可應用於淨化油汙染的土壤。
從北海道油田的遺跡A和B這兩個地點收集土壤和植物。 使用珠式细胞破碎裝置破碎土壤樣品後，提取DNA。 使用提取的DNA作為模板，進行針對總細菌代表16S rRNA基因的微生物群落做PCR分析，且具有增幅的DNA片段進行克隆與序列分析，以研究A和B兩個產地的微生物群落構造和特性。 關於烷烴功能降解相關之功能性基因，GN-RHDα，GP-RHDα，catA和pcaH是參與多環芳烴（PAH）降解酶的基因。 選擇CYP153和alkB作為參與線性烴降的酶的基因。 並透過PCR反應去確認是否存在DNA片段的擴增，並且針對擴增的DNA片段進行克隆與序列分析和系統發生樹( phylogenetic tree )的建立。
針對六個功能性基因P450（CYP153），alkB，GN-RHDα，GP-RHDα，catA，pcaH和總細菌的代表性基因16S rRNA進行PCR分析。針對石狩油田產地（A）和無煙海灘（B）的土壤中提取的DNA中16S rRNA和P450（CYP153）出現增幅片段，但對alkB，nidA，GP-RHDα，catA，pcaH，石狩油田產地（A）和無煙海灘（B）均未出現增幅片段。 因此，我們決定純化16S rRNA和P450（CYP153）的PCR產物後進行克隆與序列分析，並以非特異性DNA增幅片段對GN-RHDα進行巢式聚合酶鏈鎖反應(nested Polymerase Chain Reaction)。
通過針對16S rRNA基因和GN-RHDα的克隆與序列分析，檢出了假單胞菌屬的細菌(Pseudomonas)，被判斷是存在北海道油田產地土壤中的烴降解細菌的主要種類。 將來，將這些具有降解烷烴能力的菌群分離出來，並可應用於石油污染的地區的淨化。

Hydrocarbons are known to be stable in the environment and less susceptible to biodegradation. Furthermore, since it is carcinogenic and accumulates in the body, there is concern about the danger to human health. There are several methods for removing hydrocarbons from the environment, and among them, bioremediation, which is a repair method using microorganisms, is said to be environmentally friendly, low cost, and cost effective. In this study, using DNA extracted from the soil of the remains of the Hokkaido oil field, which contains high concentrations of alkanes and PAH, a microbial community having n-alkane and PAH-degrading bacteria that can be used to purify the soil where oil leaked.
Soil and plants were collected from two locations, A and B, which are the remains of the Hokkaido oil field. Soil samples were crushed using a multi-bead shocker and then DNA was extracted. Using the extracted DNA as a template, PCR targeting the 16S rRNA gene possessed by all bacteria was performed, and clonal analysis was performed on the amplified DNA to investigate the microbial community structures at two locations, A and B. Regarding functional genes, GN-RHDɑ, GP-RHDα and catA, pcaH are genes encoding enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation. CYP153 and alkB were selected as genes encoding enzymes involved in the degradation of linear hydrocarbons. The presence or absence of amplification of the target DNA fragment was confirmed by PCR, and clone analysis and phylogenetic tree were created for the amplified DNA fragment.
Bacteria of the genus Pseudomonas were detected by clonal analysis targeting the 16S rRNA gene and GN-RHDα, and were considered to belong to the major group as hydrocarbon-degrading bacteria inhabiting the soil of the remains of the Hokkaido oil field. In the future, these hydrocarbon-degrading bacteria will be isolated and used in petroleum-contaminated areas.

摘要 I
Abstract III
致謝 VI
目錄 VII
圖目錄 X
表目錄 XIII
第一章 緒論 1
1.1研究緣起 2
1.2 研究目的 3
第二章 文獻回顧 4
2.1 烷烴分解菌之特性 4
2.1.1 烷烴微生物之降解特性 5
2.1.2 具有烷烴降解基因的微生物的類型 5
2.1.3降解烷烴的細菌的特性 6
2.2 生活在海水中的烷烴降解細菌 7
2.3 生活在受汙染土壤中的碳氰化物降解細菌 10
2.4 生物修復(Bioremediation) 13
2.4.1 單獨透過微生物進行的生物修復 14
2.4.2 與植物相互作用下進行生物修復 15
2.5文獻回顧總結 17
第三章 研究方法及步驟 19
3.1 研究方法 20
3.2 實驗材料與設備 21
3.2.1 試劑一覽表 21
3.2.2 設備與材料一覽表 22
3.3採樣 22
3.4 DNA提取 22
3.5 PCR 23
3.5.1通常 PCR 23
3.5.2 巢式聚合酶鏈式反應(Nested PCR) 24
3.5.3 巢式聚合酶鍊式反應PCR引物設計(格蘭氏陰性細菌) 25
3.6 PCR產物的純化 27
3.7通過電泳確認擴增的DNA片段 27
3.8 克隆與序列分析 28
3.8.1連接反應(Ligation) 29
3.8.2轉化(Transformation) 29
3.8.3液體LB培養基/液體培養物的設備 30
3.8.4質體提取(Plsmid Extraction) 30
3.8.5限制性内切酶處理 31
3.8.6序列分析 32
3.8.7波形檢查和嵌合檢查 32
3.8.8同源性分析 33
3.8.9系統發生樹建立 33
第四章 結果與討論 33
4.1 PCR分析 34
4.1.1 PCR分析 34
4.1.2巢式PCR 35
4.2所有細菌中16SrRNA基因分析 36
4.2.1 16S rRNA克隆與序列分析 40
4.2.2 16S rRNA同源性搜索 42
4.2.3 16S rRNA 系統發生樹的建立 45
4.3分析烷烴降解細菌的機能性基因P450(CYP153) 46
4.3.1 克隆和序列分析P450(CYP153) 47
4.3.2同源性搜索P450(CYP153) 48
4.3.3系統發生樹的建立P450(CYP153) 51
4.4分析PAH降解細菌的機能性基因 52
4.4.1 克隆和序列分析(GN RHDα) 54
4.4.2同源性搜索(GN RHDα) 56
4.3.3系統發生樹的建立(GN RHDα) 61
第五章 總結與建議 62

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