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|Title:||土壤脫氮菌親緣關係之分析及特殊 DNA 探針之篩選|
Phylogenetic Analysis and Specific DNA Probe Selection of Soil Denitrifiers
|Publication Year :||1998|
|Abstract:||本實驗以 26 株細菌（包括 21 株脫氮菌、 4 株腸類菌及 1 株非脫氮菌）為材料，利用 PCR ( polymerase chain reaction ）方法區分其親緣關係。實驗中分別以7 條隨機引子（ random primer ）、 nosZ 基因的3條引子及 REP ( repetitive extrogenic palindromic ）序列的2條引子來分析，並加以比較。由於菌種歧異度很大，此法在此並不能成為完全適用的分類方法，需要加以挑選適當的引子來應用。不過， Alcaligenes faecalis subsp . faecalis 的 9 株菌便可利用引子 96 來區分。而且由此法找出有些菌種特殊的 PCR 產物片段，或可視為這些菌種的 DNA 標誌 ( marker ）。
其中一株土壤細菌 Thiosphaera pantotrqpha 可同時進行異營性硝化及有氧性脫氮作用，引起我們探討的興趣。於是，將 Thiosphaera pantotropha 的基因體 DNA 隨機選殖，以非放射性標定法標定核酸片段製成探針，和 26 株菌株進行菌落雜交，篩選對 Thiosphaera pantotropha 具有專一性的探針，以便偵測土壤中此菌的存在與否。結果經由 BamHI、 EcoRI 、HindIII切割出的片段較易偵測出 Alcaligenes 這一屬的菌株。此外，利用 PCR 方法放大 Pseudomonas stutzeri 的nosZ 基因非保留性片段，可以得到對本身菌種具專一性之探針。
Phylogenetic relationship among 26 bacteria including 21 denitrifiers, 4 enterobacteria and 1 nondenitrifier were distinguished by PCR methods that rely on different amplification priming strategies: RAPDs (random amplified polymorphic DNAs) PCR, REP ( repetitive extragenic palindromic ) PCR and nosZ conserved region PCR. The PCR products by 7 random primers, 2 REP primers and 3 nosZ primers were analyzed and compared to one another. However, the bacteria we used have large diversity in taxanomy, the results of the PCR assays were not fully consistent with their classification, and we may choose appropriate primers for analysis. However primer 96 could be used to distinguish 9 strains of Alcaligenes faecalis subsp. faecalis. And some genus-specific or species- specific DNA fragments in PCR products could be selected as genetic markers for identification of these organisms.
We were interested in one of the soil bacteria-Thiosphaera pantotropha which has the capacity of aerobic denitrification and heterotrophic nitrification simutaneously. Thus, the genomic DNA of T pantotropha was digested by restriction enzymes and cloned randomly. The isolated DNA fragements were labeled by using nonradioactive label system, and hybridization with 26 soil bacteria to select specific probes for T pantotropha were performed. However, it is easily to detect Alcaligenes sp. by DNA fragments digested with BamHI, EcoRI and HindIII. Besides, the probe prepared from amplifying the unconserved region of the nosZ gene of Pseudomonas stutzeri by PCR method had high specificity to Ps. stutzeri itself.
|Appears in Collections:||植物科學研究所|
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