DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil.
DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil.
复制标题
DOI：
10.1016/s0378-1097(04)00045-x
复制
发表时间：
2004-03
影响因子：
2.1
通讯作者：
Takeshi Watanabe;S. Asakawa;Asumi Nakamura;K. Nagaoka;M. Kimura
中科院分区：
文献类型：
--
作者：
Takeshi Watanabe;S. Asakawa;Asumi Nakamura;K. Nagaoka;M. Kimura
提出了一种变性梯度凝胶电泳（DGGE）分析水田土壤中产甲烷古菌群落16S rDNA的方法。首先利用13株产甲烷古菌纯培养菌株的基因组dna，采用聚合酶链反应和DGGE技术对5个产甲烷古菌16S rDNA特异性引物进行了鉴定。5对引物中的2对（0348aF-GC和0691R； 0357F-GC和0691R）可以进行DGGE分析，但部分非产甲烷古菌的16S rDNA被0348aF-GC和0691R扩增。对这两对引物进行了进一步评价，可用于日本稻田土壤产甲烷古菌群落分析。这两对引物在DGGE分析中获得了良好的分离性和图谱质量。从DGGE凝胶中共切除41个DNA片段并测定其序列。所有碎片均属于产甲烷古菌。上述结果表明，利用引物对0357F-GC和0691R进行DGGE分析的方法适用于稻田土壤中产甲烷古菌群落的研究。
A denaturing gradient gel electrophoresis (DGGE) method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil is presented. Five specific primers for 16S rDNA of methanogenic archaea, which were modified from the primers for archaea, were first evaluated by polymerase chain reaction and DGGE using genomic DNAs of 13 pure culture strains of methanogenic archaea. The DGGE analysis was possible with two primer pairs (0348aF-GC and 0691R; 0357F-GC and 0691R) of the five pairs tested although 16S rDNA of some non-methanogenic archaea was amplified with 0348aF-GC and 0691R. These two primer pairs were further evaluated for use in analysis of methanogenic archaeal community in Japanese paddy field soil. Good separation and quality of patterns were obtained in DGGE analysis with both primer pairs. A total of 41 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic archaea. These results indicate that the procedure of DGGE analysis with the primer pair 0357F-GC and 0691R is suitable for investigating methanogenic archaeal community in paddy field soil.