台灣溫泉區和石灰華環境微生物生態之調查和其生物資源利用之研究

Abstract

本研究第一部分為利用16S rRNA之序列演化和螢光原位雜交技術 (fluorescence in situ hybridization, FISH) 針對陽明山馬槽之露頭溫泉樣本進行分析。經由電腦模擬得出最適切位之 AciI, BstUI 和 RsaI三個限制酵素，192個16S rRNA clone經由 RFLP 得出九個分群。經由資料庫比對搜尋，發現其中clone M70和M6和古生菌 Crenarchaeota 門當中之Sulfolobus和Caldisphaera 相似度最大。其他之分群亦分別和資料庫當中分佈在東北亞區域熱泉或嗜熱極地環境uncultured 或 unidentified之古生菌有高度之序列相似度，顯示台灣和日本、中國和菲律賓等地之極地環境當中微生物族群狀況之相似性。在 FISH 的部分，土壤和水樣利用DAPI (4’, 6-diamidino-2-phenylindole) 和螢光標定之ARCH915, EUB338, EUK516 和 SRB385針對古生菌、真細菌、真核生物和嗜硫菌之專一性引子進行雜交，於螢光顯微鏡下觀察結果顯示分別有15.69 % 和 7.16 % 之 DAPI-stained cell可和 ARC915 和 SRB385進行雜交，顯示本區域存在相當豐富之極域微生物。第二部分為分析太魯閣峽谷內三個地點長春祠、白楊步道外及燕子口三種石灰華樣品微生物。從微生物多樣性分析，結果為長春祠、白楊步道外及燕子口三者之多樣性分別為 0.808，0.864，0.866，均勻度為 0.768，0.9，0.916，顯示長春祠的石灰華微生物多樣性最低。分析 381個16S rRNA clone,Proteobacteria 在三個石灰華樣本中之微生物中佔有25–30 %；第二多的微生物則是光合細菌，佔有16–28 %。另外最常在三個樣本中出現的菌相有 Acidobacteria, agricultural soil bacterium, verrucomicrobia 和 firmicutes等。第三部分自馬槽溫泉區分離株 #33-9選殖出α-glucosidase 並以市售套組表現蛋白質。以SDS-PAGE 顯示其為一分子量約 60 kD之蛋白質。另取 100 ul 之蛋白質產品以 p-nitrophenyl glucopyranoside 分析其活性，發現其最佳作用溫度為 60–70 °C，最高活性介於 pH 2–3。

The using of molecular methods has advanced our visions on environmental microbiology in recent years. Numerous breakthroughs and discoveries have been made on microbial community, ecological revolution, bio-adaptation and genetics bio-resources in extreme environments. In the first part of this study, archaeal community composition of Yangmingshan National Park was investigated by 16S rRNA and fluorescence in situ hybridization (FISH). Tetrameric restriction enzyme (TRE) was used in digestion and differentiation in the restriction fragment length polymorphism (RFLP) fragments, and AciI, BstUI and RsaI were shown to be the optimal TREs for TRE-RFLP. Nine clones were obtained in the studies, with clones M70 and M6 being found to be phylogenetically affiliated to Sulfolobus and Caldisphaera in domain Crenarchaeota, respectively, whereas seven other clones were found to be affiliated to uncultured and unidentified archaeon isolated from thermoacidic environments. Using FISH, soil and water region cells were hybridized with DAPI (4’, 6-diamidino-2-phenylindole) and specific fluorescently labeled probes. 15.69 % and 7.16 % of the DAPI-stained cells hybridized with universal archaeal probe ARC915 and sulfate-reducing bacterial probe SRB385, respectively. In second part, microbial diversity of tufa found in Taroko National Park was investigated using 16S rRNA cloning and FISH. Diversity index of Eternal Spring Shrine, Baiyang walkway and the Swallow valley showed to be 0.808, 0.864 and 0.866, respectively. The Evenness index also showed 0.768, 0.9 and 0.916 at those three sites. This implied low microbial diversity in E. S. Shrine. Eleven 16S rRNA phylotypes and 37 genus and group of bacteria were identified. Of total 381 clones isolated, proteobacteria occupied 25–30 % whereas cyanobacteria dominated 16–28 % in total microbial population of above three sites. Acidobacteria, agricultural soil bacterium, verrucomicrobia and firmicutes were generally distributed in these sampling sites. In the third part, α-glucosidase was cloned from isolate #33-9 and the protein was expressed using commercial cell-free transcription and translation kit. The molecular weight of the translated product is approximately 60 kD as shown by SDS-PAGE. The product was assayed for activity using p-nitrophenyl-glucopyranoside. The optimum temperature and pH for the enzyme were shown to be 60–70 °C and 2–3, respectively.