摘要
The polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method reported by Schwieger and Tebbe (1998) was used to analyze the diversity of methanogens inhabiting the rumen. Partial 16S rRNA gene fragments were amplified from DNA extracted from rumen contents by PCR with archaea-specific primers, Ar1000F and Ar1500R, or methanogen-specific primers, M301F and M915R, with one primer phosphorylated at the 5′ end. The amplified DNA fragments were analyzed by SSCP gel electrophoresis after the phosphorylated strands of the PCR products were digested with λ exonuclease. When we analyzed samples collected from the six Holstein cows used in a previous study, in which cows were given feed with or without -cyclodextrin-horseradish oil complex (CD-HR), nine and six bands were identified in the profiles generated by PCR products amplified with archaea-specific and methanogen-specific primers, respectively. While dendrogram analysis based on SSCP gel profiles found that the methanogens from each rumen showed a particular composition of methanogens, the profiles of the methanogens isolated from two of three cows fed with CD-HR fell into the same branch in the dendrogram constructed from the profiles. Therefore, this study demonstrates the potential of the PCR-SSCP method in the methanogenic community analysis of the rumen and in investigating changes in the methanogenic community due to the addition of CD-HR to the rumen.
摘要译文
聚合酶链反应单链构象多态性（PCR-SSCP）方法报道Schwieger和特伯（1998）是用来分析甲烷居住在瘤胃的多样性。部分16S rRNA基因片段从DNA的瘤胃内容物提取的PCR与古菌特异性引物，Ar1000F和Ar1500R，或甲烷特异性引物，M301F和M915R放大，用一个引物磷酸化的5核酸外切酶。当我们分析了来自于先前的研究中所使用的六个荷斯坦奶牛收集的样品，其中奶牛给予饲料含或不含环糊精 - 辣根油复合物（CD-HR）九六阶鉴定中通过分别扩增古菌特异性和甲烷特异性引物，PCR产物产生的轮廓。而聚类分析根据SSCP凝胶剖面发现从每个瘤胃的甲烷表明甲烷的特定组合物，从二三奶牛带CD-HR馈送分离的甲烷的轮廓落入在从剖面构造的树形同一分支上。因此，monstrates在瘤胃中的甲烷生成社区分析的PCR-SSCP法及在调查由于加入的CD-HR的瘤胃中的变化甲烷社区的潜力。
Noriko TATSUOKA[1]; Nazimuddin MOHAMMED[1]; Makoto MITSUMORI[2,*]; Kiyoshi TAJIMA[2]; Kozo HARA[1]; Mitsunori KURIHARA[2] and Hisao ITABASHI[3];. Analysis of methanogens in the bovine rumen by polymerase chain reaction single-strand conformation polymorphism[J]. Animal Science Journal, 2007,78(5): 512-518