Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/27999
標題: Gordonia菌屬放線菌之分子偵測、分類及鑑定
Molecular Detection, Classification and Identification of Members of the Genus Gordonia
作者: 沈佛亭
Shen, Fo-Ting
關鍵字: Gordonia
戈登氏菌
molecular detection
classification
identification
bioremediation
分子偵測
分類
鑑定
生物復育
出版社: 土壤環境科學系所
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摘要: The importance of the emerging genus Gordonia in industrial and environmental biotechnology is well evidenced from recently increasing publications and patents. But, due to the limitations with isolation or detection techniques, explorations of the valuable Gordonia members are restricted. This motivated us to design genus-specific oligonucleotides primer pairs which can assist in rapid detection of species of the genus Gordonia by means of PCR-specific amplification. Members of the metabolically diverse genus Gordonia were isolated from various biotopes including pristine and polluted sites around Taiwan. Identification, comparison and diversity assessment based on gyrB gene were carried out using a newly developed primer pair for gyrB gene and 16S rRNA gene was also sequenced for comparison. The Gordonia specific 16S rDNA fragment (829 bp or 436 bp) was successfully amplified for all the reference Gordonia species with the designed primer pair G268F/G1096R or G699F/G1134R whereas, no amplification was noted for closely-related species from other genera. The genus specificity was validated with 47 strains including wild type isolates. Preliminary screening of soil samples with Gordonia-specific primers was successful in the rapid detection of the presence of 36 Gordonia wild-type isolates. A 1.2 kb fragment of the gyrB gene of 17 Gordonia strains including type strains were determined by direct sequencing of PCR amplified fragments. Total 25 strains (8 were retrieved from public database) of genus Gordonia form a distinct phyletic line in the gyrB-based tree and are separated from other closely-related species of genera to suborder Corynebacterineae. Sequence similarity of gyrB sequence from twelve Gordonia type strains ranged from 79.3% to 97.2%, corresponding to 270-41 nucleotide differences while there was only 0.3-3.8% difference in 16S rRNA gene sequence similarity at the interspecies level. Phylogenetic analysis based on gyrB gene is consistent with those of 16S rDNA tree and provided a better discrimination within the species of Gordonia compared to 16S rRNA gene. The present study demonstrates that gyrB gene analysis will aid in describing novel species belonging to the genus Gordonia. The detection and analyses of Gordonia populations within complex microbial communities were achieved precisely and rapidly by PCR-DGGE method and provide better assessment of the species diversity of Gordonia in the environment.
Gordonia (戈登氏菌)菌屬是近年來逐漸受到環境污染處理的重視,且廣泛被研究的一屬放線菌。此屬之微生物具有降解多環類芳香族碳氫化合物的特性,在生物復育上扮演重要角色。篩選Gordonia分離株不但可開發新放線菌資源,並且了解此類放線菌之分布及在生態中的角色,此外研究Gordonia屬內菌種之親緣關係,有助於了解各菌種之演化地位,並提供土壤中可靠且快速之鑑定結果。本研究為加速環境中Gordonia菌種之篩選,利用16S rRNA基因作為標的,設計Gordonia菌屬特異性引子,並配合聚合酶連鎖反應(PCR)之快速偵測技術鑑定此類放線菌。此外為提高對Gordonia 16S rDNA序列相似度極高菌種間(大於99%)之鑑別能力,本研究首度設計針對Gordonia放線菌gyrB基因之PCR增殖及定序引子,探討演化標誌基因16S rRNA與gyrB基因在Gordonia菌種間與不同分離株間之差異性。研究結果顯示,利用G268F/G1096R及G699F/G1134R兩組Gordonia菌屬特異性引子,可有效區別Gordonia菌屬與其他放線菌菌屬,並且利用此偵測技術已從台灣分離出36株Gordonia分離株。經多序列分析比對軟體計算並比較gyrB基因序列,可知Gordonia菌屬與7種鄰近放線菌的gyrB基因序列相似度介於72.8-82.1%,而在16S rDNA序列相似度則介於90.0-95.4%,gyrB基因可明顯區分Gordonia菌屬與其他屬之放線菌。在Gordonia菌種間之差異研究中則發現12種Gordonia gyrB基因序列相似度介於79.3%-97.2%,16S rDNA的序列相似度為96.2%-99.7%,可知Gordonia放線菌的16S rDNA保留性極高,較難利用此基因序列進行菌種間之區分。gyrB基因之變異程度無論是在菌屬、菌種甚至各分離株間均較16S rRNA基因大。本研究之結果將可利用gyrB基因作為Gordonia菌種快速鑑定之依據,若兩菌株之16S rDNA序列相似度大於99.7%、gyrB基因相似度大於97.2%,則判定兩菌株為相同菌種。本研究利用上述兩基因序列相似度初步判定4株本土Gordonia新菌種之分類地位,其中分離株CC-AB07經由生理生化特性及化學組成分析,已確認為本土Gordonia新菌種。此外利用菌屬特異性引子配合變性梯度凝膠電泳(DGGE),成功應用至泡沫污水中Gordonia族群之分析,未來將探討Gordonia在環境中之族群多樣性。
URI: http://hdl.handle.net/11455/27999
其他識別: U0005-0607200616503000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0607200616503000
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