Analysis of methanogenic archaeal community predominated in paddy field soil

本研究的目的是建立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PCR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O348aF-GC和0691R扩增。对这两对引物进行了进一步的评价，并将其应用于日本千果和安条两个稻田土壤中产甲烷古菌群落的分析。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凝胶中共提取41个DNA片段并测定其序列。所有片段均属于产甲烷古生菌，分别属于甲烷微生物门、甲烷化石门和“水稻簇”。结果表明，0357F-GC和0691R引物对DGGE分析方法适用于稻田土壤中产甲烷古菌群落的研究。目前，我们正在用这种方法对上述两个地区的群落进行季节动态分析。

The objective of the present research project was to establish a denaturing gradient gel electrophoresis (DGGE) method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil. Five specific primers for 16S rDNA of methanogenic archaea, which were modified from the primers for archaea, were first evaluated by PCR 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 nonmethanogenic archaea was amplified with O348aF-GC and 0691R. These two primer pairs were further evaluated for use in analysis of methanogenic archaeal communities in soil of two paddy fields in Chikugo and Anjo, Japan. Good separation and quality of patterns were obtained in DGGE analysis with the both primer pairs. Totally 41 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic archaea and belonged to Methanomicrobiales, Methanosarcinales and "Rice clusters". These results indicate that the procedure of DGGE analysis with the primer pair of 0357F-GC and 0691R is suitable for investigating methanogenic archaeal community in paddy field soil. Now we are analyzing seasonal dynamics of their communities in the above two fields by this method.