Analysis of methanogenic archaeal community predominated in paddy field soil

本研究旨在建立稻田土壤产甲烷古菌16 SrDNA的变性梯度凝胶电泳（DGGE）分析方法。以13株产甲烷古菌纯培养菌株的基因组DNA为材料，利用PCR和DGGE技术，首次对5条产甲烷古菌16 SrDNA特异性引物进行了评价。在所测试的5对引物中，有2对引物（0348 aF-GC和0691 R; 0357 F-GC和0691 R）可以进行DGGE分析，但O348 aF-GC和0691 R可以扩增一些非产甲烷古菌的16 SrDNA。这两个引物对进一步评估用于分析产甲烷古菌群落在两个稻田在筑后和安城，日本。用这两对引物进行DGGE分析，获得了良好的分离效果和高质量的图谱。从DGGE凝胶上切下41个DNA片段，并测定其序列。所有片段均属于产甲烷古菌，属于甲烷微生物目、甲烷微生物目和“稻簇”。以上结果表明，以0357 F-GC和0691 R为引物的DGGE分析程序适用于稻田土壤产甲烷古菌群落的研究。目前，我们正利用这一方法分析这两个领域的群落季节动态。

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.