臺灣博碩士論文加值系統

紫色非硫菌為一群行厭氧光合作用的微生物，在不同的環境可進行不同之代謝模式包含：光合自營、光合異營、化學異營及化學自營。由於這類細菌的特殊生理特性，近年來受到科學家高度的重視。它們廣布於自然界，包含湖泊、水塘、海洋及一些水田等等。
由北台灣淡鹽水體當中篩出39株菌種，探討它們外形上的多樣性：菌體形態、菌液顏色與菌落外觀，以及16S-23S ribosomal intergenic spacer region(ISR)序列的多樣性並加以分類。首先，將它們分成七種不同的菌體形態：球狀、卵形、環形、馬蹄形、及三種不同大小的桿狀，菌液顏色有褐色、紫色、粉紅色及咖啡色等；而菌落外觀為圓形，表面光滑，邊緣平整。再分別依每個不同的菌體觀察其菌液顏色及菌落形態的不同，共分成26類。將26類的代表菌株進行16S rRNA序列的分析，經親緣關係比對結果皆大於99%，表示這26株菌種皆為紫色非硫菌，主要屬於Rhodobacter sphaeroides、Rhodovulum sulfidophilum及Rhodopseudomonas sp.三個屬，且這三個屬的菌株所呈現的外觀形態有高度的多樣性。另一方面，利用PCR增幅此39株菌種的16S-23S ISR片段，再以PvuII、AluI及HaeIII三種酵素作用，會呈現22種RFLP圖譜，因此可分成22群。其中比對結果為Rba. sphaeroides及Rhodovulum sp. strain MB263分別產生7種與2種不同的圖譜，表示在這兩株菌的16S-23S ISR區域具有多樣性或者資料庫無法比對出此圖譜所相對應的菌種，而其他16S rRNA基因序列的比對結果相同的菌株皆能與圖譜的分群結果一致。因此利用16S-23S ISR-RFLP可輔助16S rRNA基因序列比對，建立出一個分類並鑑定紫色非硫菌的系統。

Purple non-sulfur bacteria are a group of extraordinary metabolic
diverse bacteria. They can grow with photoautotroph, photoheterotroph, chemoheterotroph or chemoautotroph. Under various conditions, they can enjoy exceptional flexibility within each of these modes of metabolism. Due to the special physical characteristics properties, they had attracted scientist’s attention in resent years. These bacterias are widely distributed in nature such as lakes, water ponds, seas, or farm, etc. We isolated 39 strains bacteria from different aqueous phase of northern Taiwan. We discussed the diversity of their morphology and genetic diversity of their 16S-23S ribosomal intergenic spacer region(ISR) . First, dependent on cell shapes, we classed these strains into seven types: coccus, oval, ring, horse's hoof, and three size rods. Colors of their cell suspension are brown, pink, purple and red-brown, etc. Their cultural characteristics almost are circular forms , convex elevation and entire margins. Then, according to above characteristics, we classed 39 strains in to twenty-six clusters. Taking 16S rRNA sequence of these 26 representatives to blast with DataBank , the similarity all are larger than 99%，which reveals they were related to Rhodobacter sphaeroides, Rhodovulum sulfidophilum and Rhodopseudomonas sp. and revealed there were highly diversity of their morphology . In other hand, the16S-23S ISR of 39 strains were amplified by PCR and digested with PvuII, AluI, HaeIII, resulting in 22 RFLP profiles. This results showed that 39 purple nonsulfur bacterias could be classed into 22 groups. 16S-23S ISR-RFLP revealed that there was a genotype diversity within Rba. sphaeroides and Rhodovulum sp. strain MB263,which there were different 7 and 2 RFLP profiles respectively. This result showed that there was genotype diversity of 16S-23S ISR with both species ,or may that there was not enough database of 16S rRNA sequence in GenBank to blast accurately to the species. Hence, analysis of 16S-23S ISR-RFLP was more accurate than analysis of 16S rRNA sequences for identification of purple non-sulfur bacteria. By this method, 16S-23S ISR-RFLP, we can set up a characterization system of purple non-sulfur bacteria.

目錄
中文摘要................................................................................................I
英文摘要..............................................................................................III
目次......................................................................................................V
表次....................................................................................................VII
圖次...................................................................................................VIII
前言.......................................................................................................1
第一節、 紫色非硫菌之簡介................................................................1
第二節、 紫色非硫菌之應用................................................................2
第三節、 紫色非硫菌之生長條件與培養..............................................2
第四節、 紫色非硫菌之分類與多樣性…………………………………..6
第五節、 16S-23S ribosomal intergenic spacer region簡介與應用.........................................................................................8
第六節、 微生物產生PHA基因之研究..............................................10
材料方法..............................................................................................12
第一節、 材料與方法........................................................................12
第二節、 儀器設備............................................................................21
結果.....................................................................................................22
討論.....................................................................................................36
結論.....................................................................................................44
參考文獻..............................................................................................45

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