Analysis of ecology of rumen bacterial populations by molecular biological techniques

通过分子生物学技术分析瘤胃细菌种群的生态学

• 批准号: 11660284
• 负责人: KOBAYASHI Yasuo
• 金额: $ 1.79万
• 依托单位: KOKKAIDO UNIVERSITY (2000)Mie University (1999)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660284/
• 关键词: Rumen; Cellulolytic bacteria; Competitive PCR; Sheep; DNA; DNA抽出; 定量; 特異プライマー; 付着

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.

本研究采用竞争性PCR和FISH两种新建立的分子生物学技术，对瘤胃中三种具有代表性的纤维素分解细菌进行了生态学分析。通过技术的开发和应用，取得了以下结果：使用针对目标菌种的16SrDNA序列设计的引物允许对相应菌种进行特异性扩增。所建立的cPCR检测方法在纯培养和混合培养水平上具有较高的定量，然后检测值与添加已知数量的目标细菌呈线性响应。测定值重复性好。试验结果表明，琥珀酸梭菌在绵羊瘤胃中比两种瘤胃球菌更占优势，特别是在饲喂高粗饲料时。后肠中琥珀酸梭菌的含量与瘤胃中相似。这些都表明了这种细菌对胃肠道中纤维素分解的主要贡献。FISH用于在显微镜下观察目标细菌种类。建立细菌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检测目标细菌。

项目成果

小林泰男: "Development of a competitive PCR assay for the ruminal Bacterium Bfibnluems cB156 and its use for tracking an cB156 derived necombinant"FEMS Microbiology Letters. 188. 185-190 (2000)

Yasuo Kobayashi：“瘤胃细菌 Bfibnluems cB156 的竞争性 PCR 测定的开发及其用于追踪 cB156 衍生重组体的用途”FEMS 微生物学快报 188. 185-190 (2000)。

小林 泰男: "Application of Molecular Biology to Rumen Microbes-Review-"Asian-Aust: Journal of Animal Sciences. 12・1. 77-83 (1999)

Yasuo Kobayashi：“分子生物学在瘤胃微生物中的应用-评论-”亚洲-澳大利亚：动物科学杂志 12・1（1999）。

小林泰男: "Application of molecular biology to rumen microbes-Review-"Asian-Australasian Journal of Animal Sciences. 12. 77-83 (1999)

Yasuo Kobayashi：“分子生物学在瘤胃微生物中的应用-评论-”亚洲-澳大利亚动物科学杂志 12. 77-83 (1999)。

Kobayashi, Y.et al.: "Development of a competitive PCR assay for the ruminal bacterium B.fibrisolvens OB156 and its use for tracking an OB156-derived recombinant."FEMS Microbiology Letters. 188. 185-190 (2000)

Kobayashi, Y. 等人：“针对瘤胃细菌 B.fibrisolvens OB156 的竞争性 PCR 测定的开发及其用于追踪 OB156 衍生重组体的用途。”FEMS 微生物学快报。

小林泰男: "セルロースの事典"朝倉書店. 580 (2000)

小林康雄：《纤维素百科全书》朝仓书店 580 (2000)。