Analysis of ecology of rumen bacterial populations by molecular biological techniques

本研究尝试用竞争PCR和FISH这两种最新建立的分子生物学技术对瘤胃内三种具有代表性的纤维分解菌进行生态学分析。利用针对目标菌种的16SrDNA序列设计的引物可以对相应菌种进行特异扩增。建立的CPCR方法在纯培养和混合培养水平上都是高度定量的，然后测定值与已知的目标细菌数量的添加呈线性关系。测定值的重现性令人满意。结果表明，在绵羊瘤胃中，产琥珀酸杆菌比两种反胃球菌更具优势，尤其是在饲喂高粗饲料时。产琥珀酸假单胞菌在后肠中的含量水平与瘤胃相似。这些都表明这种细菌对胃肠道中的纤维分解做出了主要贡献。FISH是为了在显微镜下显示目标细菌物种而开发的。在建立了细菌RNA与荧光标记DNA探针杂交的条件后，在纯培养和混合培养水平上对目标细菌进行了检测。

The present study was attempted to analyze ecology of three representative cellulolytic bacterial species in the rumen by newly established molecular biological techniques that were competitive PCR and FISH.The results obtained througt development of the techniques and their application are follows.1. Primers designed by using 16SrDNA sequences specific to the target bacterial species allowed specific amplification of the corresponding species. The developed cPCR assays were highly quantitative at pure and mixed culture levels, then the assay values linearly responded to the addition of known amount of target bacteria. The assay values were satisfactorily reproducible.2. In application of the assays, it was revealed that F.succinogenes wa more dominant in sheep rumen than two ruminococcal species, especially when a high roughage diet was given. F.succinogenes was quantitated in the hindgut at a level similar to that in the rumen. These are indicative of major contribution of this bacterial species to cellulolysis in the gastrointestinal tract.3. FISH was developed to visualize the target bacterial species under microscopy. After condition of hybridization of the bacterial RNA with a fluorescence-labelled DNA probe was established, the target bacteria was detected at pure and mixed culture levels.