臺灣博碩士論文加值系統

鏈黴菌屬，是新的次級代謝物的重要的來源，其生物活性範圍包括抗菌、抗癌以及免疫抑制活性，在製藥工業應用及人類健康照護非常重要。台灣鋏蠓於台中市大坑地區肆虐，使本校的師生與當地居民也不堪其擾。過去研究顯示台灣鋏蠓的密度與環境密切相關，例如：台灣鋏蠓幼蟲的食物藻類的孳生。鮑氏不動桿菌為臨床常見院內感染病原菌，常於免疫力低下的患者造成菌血症、泌尿道感染、腦膜炎及肺炎等。植物的病害造成農產品巨大損失，例如 Fusarium oxyporum 及 Rhizoctonia solani 引發的疾病在全球造成巨大經濟損失。先前本實驗室於大坑地區分離三株 Streptomyces，可抑制細菌、綠藻/藍綠藻生長，本研究主要進行此三株 Streptomyces 特性分析，並評估此三株 Streptomyces 抑菌、抑藻及抑真菌實際應用潛力。本研究進行以下實驗：以 actinomycete 16S rRNA 及 RNA polymerase -subunit (rpo) 基因片段進行 PCR 增幅並鑑定 Streptomyces 的身份；測試 Streptomyces 上清液抑菌、抑藻及抑真菌效果；測試 Streptomyces 上清液是否會影響台灣鋏蠓幼蟲生長；測試 S1 萃取液抑菌、抑藻及抑真菌效果；測試 S1 上清液是否可抑制臨床多重抗藥性鮑氏不動桿菌生長；使用 S1 萃取液合併 Imipenem 測試對臨床多重抗藥性鮑氏不動桿菌的協同效果。實驗結果顯示：Streptomyces S1 之 16S rRNA 序列與 S. manipurensis 有 100% 相似度，D7 與 Uncultured Streptomyces sp. clone T38 有 99% 相似度，D9 與 Uncultured actinobacterium isolate 有 99% 相似度；rpo 序列至 Blastx 比對，顯示 S1 與 Streptomyces sp. BDUSMP S05 rpo 有 96% 相似度，D7 與 Streptomyces sp. BDUSMP R07 有 94% 相似度，D9 與 Streptomyces albus 有 98% 相似度。Streptomyces S1、D7、D9 上清液可抑制革蘭氏陽性/陰性菌、綠藻/藍綠藻及真菌生長。S1、D7、D9 的上清液對台灣鋏蠓幼蟲的生長無明顯影響。S1 上清液以 30%、40% 甲醇及 SPE column 沖提後所收集的萃取液抑菌、抑藻及抑真菌效果較顯著。S1 上清液可抑制臨床多重抗藥性鮑氏不動桿菌生長。Imipenem與 S1 30%、40% methanol 萃取液合併測試時，抑制圈慢慢增加。綜合以上結果，顯示此三株 Streptomyces 皆具有抑制細菌、藻類及真菌生長的能力，但 S1、D7、D9 上清液對台灣鋏蠓幼蟲生長及發育無明顯影響，S1 萃取液與 Imipenem 合併使用可增加抑菌效果，後續進一步評估實際應用或發展為生物防治之可行性。

Streptomyces are renowned sources of novel secondary metabolites which have a range of biological activities such as antimicrobial, anticancer, and immunosuppressive activities, so the application of the pharmaceutical industry and human health care is very important. Forcipomyia taiwana was rage in the Da-Kan area of Taichung City, also caused on teachers and students of the school and the local residents it’s unbearable. Previous reports showed that the density of F. taiwana has a great relationship with the environment, such as the breeding of algae that food of F. taiwana larvae. Acinetobacter baumannii is common nosocomial which causes a wide variety of infections in immunocompromised patients, such as bacteremia, urinary tract infections, meningitis and pneumonia. Plant diseases cause huge losses of agricultural products, especially diseases caused by soil plants, Fusarium oxyporum and Rhizoctonia solani causing huge economic losses in the world. Previously, three Streptomyces isolates were isolated from the pit area and tested to have the ability to inhibit green algae, blue-green algae and bacterial growth. Characterization of the three Streptomyces were analyzed and the potential for antimicrobial, anti-algae and antifungal was evaluated. In this study: Identification of Streptomyces strains by amplification and blasting of actinomycete 16S rRNA and rpo. Test the secretion of substances have antibacterial effect by Streptomyces culture supernatant for antimicrobial, anti-algae and antifungal assay. To test the effect of Streptomyces supernatant on the growth of F. taiwana larvae. To test S1 extract for antibacterial, anti-algae and antifungal assay. S1 supernatants were tested for their ability to inhibit clinical multi-drug resistant Acinetobacter baumannii growth. Combination test with Imipenem and S1 extract against clinical isolates of multidrug resistant A. baumannii. Results: The actinomycete 16S rRNA partial sequence of S1 isolate was 100% identical with S. manipurensis, D7 isolates were 99% identical with Uncultured Streptomyces sp. clone T38, D9 isolates were 99% identical with Uncultured actinobacterium isolate. The rpo sequence of S1 isolate was 96% identical with Streptomyces sp. BDUSMP S05 RNA polymerase subunit B and D7 isolates were 94% identical with Streptomyces sp. BDUSMP R07, D9 isolates were 98% identical with Streptomyces albus. The growth of bacterial, green algae, blue-green algae and fungi were inhibited by S1, D7, D9 culture supernatants. The supernatant of S1, D7 and D9 culture had no significant effect on the growth of F. taiwana larvae. The antibacterial, anti-algae and antifungal effect of S1 supernatant extracted with 30%, 40% methanol and SPE column was most significant. The clinical isolates of multi-drug resistant A. baumannii were inhibited by S1 culture supernatants. Imipenem and S1 30%, 40% methanol extract combined test, the inhibition zone slowly increased. These results suggest that three Streptomyces isolates all had the ability to inhibit the growth of bacteria, algae and fungi, but supernatant of Streptomyces S1, D7, D9 culture had no significant effect on the growth of F. taiwana larvae, S1 extracts combined with Imipenem can increase the inhibitory effect, and should further evaluate the feasibility of practical application or development for biocontrol.

摘要............................................................................................................................................ I
Abstract ................................................................................................................................. III
目錄........................................................................................................................................... V
圖目錄................................................................................................................................... VII
表目錄.................................................................................................................................. VIII
第一章、前言............................................................................................................................ 1
第二章、文獻回顧.................................................................................................................... 5
一、 鏈黴菌
(Streptomyces)......................................................................................
6二、 鏈黴菌次級代謝物生產的影響........................................................................ 7
三、 抗菌化合物萃取................................................................................................ 9
四、 抑制試驗.......................................................................................................... 10
五、 分子技術應用於菌種鑑定.............................................................................. 19
第三章、材料與方法............................................................................................................. 22
一、 本實驗使用菌株.............................................................................................. 23
二、 菌種保存.......................................................................................................... 23
三、 儀器、試劑、藥品及酵素.............................................................................. 23
四、 細菌與真菌染色體 DNA 的製備 (extraction of chromosomal DNA by bacterial and fungal)
...................................................................................................... 27
五、 藻類染色體 DNA 的製備 (extraction of chromosomal DNA by algae)... 28
六、 以 PCR 增幅鏈黴菌之 16S rRNA 基因並鑑定 (Amplification and identification of actinomycete 16S rRNA by PCR
and sequencing in Streptomyces)29
七、 以 PCR 增幅鏈黴菌之 rpo 並鑑定 (Amplification and identification of rpo by PCR and sequencing in Streptomyces) ............................................................ 31
八 、 以 PCR 增 幅 藍 綠 藻 之 16S rRNA 並 鑑 定 (Amplification and identification of 16S rRNA by PCR and sequencing in Cyanobacteria) ................... 31
九、 PCR 增幅綠藻之 18S rRNA 並鑑定 (Amplification and identification of
18S rRNA by PCR and sequencing in Chlorophyceae)............................................... 32
十、 以 PCR 增幅真菌之 ITS1、5.8S、ITS2 gene 並鑑定 (Amplification and identification of ITS1、5.8S、ITS2 gene by
PCR and sequencing in Fungi)........... 33
十一、 以 PCR 增幅真菌之 18S 及 26S rRNA 並鑑定 (Amplification and identification of 18S and 26S rRNA by PCR and sequencing in Fungi).................... 34
十二、 以平板測試法測試 Streptomyces 上清液抑菌效果.................................... 34
十三、 以平板測試法測試 Streptomyces 上清液分泌抑菌物質最佳培養天數.... 35
十四、 測試 Streptomyces 培養液之上清液抗菌能力對溫度的感受性................ 36
十五、 測試 Streptomyces 培養液上清液抗菌能力對 Proteinase K 的感受性.. 36
十六、 以平板測試法測試 Streptomyces 上清液之抑藻效果................................ 37
十七、 以平板測試法測試 Streptomyces 上清液之抑真菌效果............................ 38
十八、 測試 Streptomyces 上清液影響台灣鋏蠓幼蟲生長之情形......................... 39
十九、 Streptomyces 培養液上清液中抗菌物質的萃取.......................................... 40
二十、 以平板測試法測試 Streptomyces 培養液之上清液抑制臨床多重抗藥性鮑氏不動桿菌效果........................................................................................................... 41
二十一、 測試 Streptomyces 萃取液合併 Imipenem/Colistin 對臨床多重抗藥性鮑氏不動桿菌的抑菌效果.......................................................................................... 41
第四章、結果.......................................................................................................................... 43
一、 Streptomyces 之 16S rRNA 序列比對結果................................................. 44
二、 Streptomyces 之 rpo 序列比對結果........................................................... 45
三、 藍綠藻之 16S rRNA 片段序列比對結果..................................................... 46
四、 綠藻之 18S rRNA 序列比對結果................................................................. 46
五、 分離之真菌的 ITS1、5.8S、ITS2 基因序列比對結果.............................. 47
六、 真菌之 18S 及 26S rRNA 基因序列比對結果.......................................... 48
七、 Streptomyces 抗菌能力測試..........................................................................
49
八、 測試 Streptomyces 抗菌物質分泌最佳培養天數........................................ 49
九、 測試 Streptomyces 培養液上清液抗菌物質對溫度的感受性.................... 50
十、 測試 Streptomyces 上清液抗菌物質對 Proteinase K 的感受性.............. 51
十一、 以 Streptomyces 上清液進行抑藻試驗........................................................ 52
十二、 以 Streptomyces 上清液測試抗真菌效果.................................................... 52
十三、 測試 Streptomyces 上清液影響台灣鋏蠓幼蟲生長之情形......................... 53
十四、 測試 Streptomyces 萃取液的抗菌及抗藻效果............................................ 53
十五、 測試 Streptomyces 上清液抑制臨床多重抗藥性鮑氏不動桿菌生長情形 56 十六、 測試 Streptomyces 萃取液合併 Imipenem/Colistin 對臨床多重抗藥性鮑氏不動桿菌的效果.......................................................................................................... 56
第五章、討論與結論.............................................................................................................. 58
參考文獻.................................................................................................................................. 64

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