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研究生:胡翔竣
研究生(外文):Hsiang-Chun Hu
論文名稱:從海洋沉積物中篩選出具生物活性之放線菌
論文名稱(外文):Isolation of bioactive actinomycetes from marine sediments
指導教授:郭傑民
指導教授(外文):Jimmy Kuo
學位類別:碩士
校院名稱:國立東華大學
系所名稱:海洋生物研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:104
論文頁數:75
中文關鍵詞:海洋沉積物海洋放線菌抗菌活性細胞毒殺活性宏基因體分析454焦磷酸定序
外文關鍵詞:marine sedimentsActinobacteriaantimicrobial activitycytotoxicity activitymetagenomic analysis454 pyrosequencing
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海洋放線菌廣泛分布於不同的海洋環境中,且是生產新類型生物活性化合物的重要來源之一。本研究將從台灣西南部海域採集海洋沉積物,採用三種不同的樣本前處理方法以及五種選擇性培養基來增加自海洋沉積物分離放線菌的比率。結果共分離出319株放線菌,在瓊脂碇抗菌活性篩選試驗中,共51株(15.99%)放線菌對兩株病原菌(Bacillus subtilis、Staphylococcus aureus)有抗菌活性。將生物活性較佳的33株放線菌以16S rDNA進行親緣關係分析,顯示其中包含7個屬:Streptomyces (23)、Saccharomonospora (5)、Micromonospora (1)、Tsukamurella (1)、Nocardiopsis (2)和Actinoalloteichus (1)。挑選抗菌活性較佳的35株放線菌放大培養,利用其粗萃物進行細胞毒殺活性試驗,獲得10個放線菌粗萃物(2K-CH04、2K-FY01、2K-CH06、2K-IB05、2K-KG14、BHD-SKE04、WD-SKE36、2K-BH13、2K-CH08及BK10-AG1)對其中幾個癌細胞株(HL-60、K-562、CCRF-CEM、MOLT4、T-47D、LNCaP與DLD-1)擁有較佳的抑制活性,可進行天然化合物純化及後續活性研究。

為了解深海沉積物的細菌多樣性,利用深度超過2000 m的2K底泥樣本,以16S rDNA放大擴增進行454焦磷酸定序,共獲得11632條16S rDNA序列,並分成1239 OTUs (97%序列相似度)。其細菌群落包含超過23個細菌門,所佔比例最高的前五名分別為變形菌門(48.98%, 450 OTUs)、擬桿菌門(10.91%, 110 OTUs)、浮黴菌門(7.41%, 146 OTUs)、綠彎菌門(6.86%, 93 OTUs)、放線菌門(4.76%, 51 OTUs)。

Marine actinomycetes, widely distributed in different marine environments and habits, are a source of new biologically active compounds. In the present study, a culture-based approach was used to isolate actinomycetes from marine sediments, collected in the southern Taiwan. To increase the abundance of actinomycetes, the extracts of marine sediments were treated with three different pretreatment methods and enriched with five different selective media using agar-based culture techniques. A total of 319 actinomycetes were isolated, among which 51 isolates (15.99%) exhibited antimicrobial activity against two indicator microbes using agar block methods. 33 actinomycetes that showed high antimicrobial activity were selected for 16S rDNA sequencing. Phylogenetic analysis of the sequences indicated these isolates belong to the following seven bacterial genera: Streptomyces (23), Saccharomonospora (5), Micromonospora (1), Tsukamurella (1), Nocardiopsis (2), and Actinoalloteichus (1). Ten actinomycetes, namely 2K-CH04, 2K-FY01, 2K-CH06, 2K-IB05, 2K-KG14, BHD-SKE04, WD-SKE36, 2K-BH13, 2K-CH08, and BK10-AG1, which have high cytotoxicity against cancer cell and antimicrobial activity were good candidates for further natural product isolation and characterization research. To understand the diversity of bacterial flora associated with marine sediments, we also investigate sample 2K by 16S rDNA tag pyrosequencing. A total of 11632 sponge-derived sequence tags were obtained and clustered to 1239 operational taxonomic units (OTUs; 97% sequence similarity). The bacterial communities associated with sample 2K were assigned to 23 bacterial phyla and revealed abundant of phyla Proteobacteria (48.98%, 450 OTUs), Bacteroidetes (10.91%, 110 OTUs), Planctomycetes (7.41%, 146 OTUs), Chloroflexi (6.86%, 93 OTUs) and Actinobacteria (4.76%, 51 OTUs).
第一章 緒論 ..................................................... 1

1.1 前言........................................................ 1
1.2 微生物與其二次代謝物.......................................... 1
1.3 海洋放線菌....................................................3
1.4 文獻回顧......................................................4
1.5 宏基因體學分析.................................................5
1.6 研究動機與目的.................................................6

第二章 實驗材料與方法..............................................11
2.1 藥品、儀器與培養基............................................11
2.1.1 藥品......................................................11
2.1.2 微生物培養基...............................................12
2.1.3 實驗儀器...................................................13
2.1.4 生物活性實驗測試菌株........................................14
2.1.5 細胞株.....................................................15
2.2 培養基配製...................................................16
2.2.1 選擇性培養基...............................................16
2.2.2 菌株培養基.................................................19
2.3 採樣及放線菌分離與培養........................................21
2.3.1 海洋沉積物採樣.............................................21
2.3.2 樣本前處理.................................................21
2.3.3 平板培養與活菌計數..........................................22
2.4 海水需求測試、放線菌之抗菌活性篩選與菌種冷凍保存..................22
2.4.1 海水需求性測試..............................................22
2.4.2 瓊脂錠(agar block)抗菌活性試驗..............................23
2.4.3 菌種冷凍保存................................................24
2.5 16S rDNA 序列鑑定放線菌之分類地位..............................24
2.5.1 萃取與純化放線菌基因體DNA....................................24
2.5.2 聚合酵素連鎖反應增幅16S rDNA 片段............................25
2.5.3 DNA 電泳分析...............................................26
2.5.4 凝膠內DNA 之純化............................................26
2.5.5 DNA 序列分析...............................................27
2.5.6 分子親緣關係分析............................................28
2.6 粗萃物抗菌活性與細胞毒殺分析....................................28
2.6.1 放線菌菌液培養與粗萃取.......................................28
2.6.2 粗萃物之細胞毒殺試驗.........................................28
2.6.3 癌細胞株生長抑制試驗.........................................29
2.7 海洋沉積物細菌群落宏觀基因體定序分析............................30
2.7.1 萃取與純化海洋沉積物細菌基因體DNA 與454 焦磷酸定序.............30
2.7.2 metagenomics 序列生物資訊學分析.............................31
2.7.3 OTU 分析...................................................31
2.7.4 稀釋性曲線分析..............................................32
2.7.6 多樣性分析.................................................32
2.7.7 分類學分析.................................................34

第三章 結果與討論.................................................39

3.1 海洋沉積物之海洋放線菌分離培養..................................39
3.2 放線菌株抗菌活性初步篩選與海水需求性測試.........................40
3.2.1 抗菌活性初步篩選............................................40
3.2.2 海水需求性測試..............................................41
3.3 抗菌活性二次篩選..............................................42
3.4 放線菌株培養液粗萃............................................43
3.5 粗萃物之細胞毒殺試驗活性.......................................43
3.6 16S rDNA 序列鑑定放線菌分析...................................44
3.7 16S rDNA 序列之分子親緣關係分析................................44
3.8 海洋沉積物細菌群落宏觀基因體定序分析結果.........................44
3.8.1 Raw read 前處理............................................45
3.8.2 OTU 分析...................................................45
3.8.3 稀釋性曲線分析..............................................45
3.8.4 多樣性分析.................................................45
3.8.5 分類學分析.................................................46

第四章 結論......................................................75

參考文獻.........................................................76
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