Cheng Yan-fen, Mao Sheng-yong, Pei Cai-xia, Liu Jian-xin, Zhu Wei-yun
Laboratory of Gastrointestinal Microbiology, Nanjing Agricultural University, Nanjing 210095, China.
Wei Sheng Wu Xue Bao. 2006 Dec;46(6):879-83.
Rumen methanogen diversity in the co-cultures with anaerobic fungi from goat rumen was analyzed. Mix-cultures of anaerobic fungi and methanogens were obtained from goat rumen using anaerobic fungal medium and the addition of penicillin and streptomycin and then subcultured 62 times by transferring cultures every 3 - 4d. Total DNA from the original rumen fluid and subcultured fungal cultures was used for PCR/DGGE and RFLP analysis. 16S rDNA of clones corresponding to representative OTUs were sequenced. Results showed that the diversity index (Shannon index) of the methanogens generated from DGGE profiles reduced from 1.32 to 0.99 from rumen fluid to fungal culture after 45 subculturing, with the lowest similarity of DGGE profiles at 34.7%. The Shannon index increased from 0.99 to 1.15 from the fungal culture after 45 subculturing to that after 62 subculturing, with the lowest similarity at 89.2% . A total of 5 OTUs were obtained from 69. clones using RFLP analysis and six clones representing the 5 OTUs respectively were sequenced. Of the 5 OTUs, three had their cloned 16S rDNA sequences most closely related to uncultured archaeal symbiont PA202 with the same similarity of 95 %, but had not closely related to any identified culturable methanogen. The rest two OTUs had their cloned 16S rDNA sequences sharing the same closest relative, uncultured rumen methanogen 956, with the same similarity of 97% .Their 16S rDNA sequences of these two OTUs also showed 97% similar to the closest identified culturable methanogen Methanobrevibacter sp. NT7. In conclusion, diverse yet unidentified rumen methanogen species exist in the co-cultures with anaerobic fungi isolated from the goat rumen.
分析了山羊瘤胃中与厌氧真菌共培养的瘤胃产甲烷菌多样性。使用厌氧真菌培养基并添加青霉素和链霉素从山羊瘤胃中获得厌氧真菌和产甲烷菌的混合培养物，然后每3 - 4天转移培养物进行62次传代培养。将原始瘤胃液和传代培养的真菌培养物的总DNA用于PCR/DGGE和RFLP分析。对与代表性OTU对应的克隆的16S rDNA进行测序。结果表明，传代培养45次后，DGGE图谱中产甲烷菌的多样性指数（香农指数）从瘤胃液的1.32降至真菌培养物的0.99，DGGE图谱的最低相似度为34.7%。传代培养45次后的真菌培养物到传代培养62次后的真菌培养物，香农指数从0.99增加到1.15，最低相似度为89.2%。使用RFLP分析从69个克隆中总共获得5个OTU，并分别对代表这5个OTU的6个克隆进行测序。在这5个OTU中，有3个克隆的16S rDNA序列与未培养的古菌共生体PA202关系最密切，相似度均为95%，但与任何已鉴定的可培养产甲烷菌关系不密切。其余两个OTU克隆的16S rDNA序列具有相同的最相近亲缘关系，即未培养的瘤胃产甲烷菌956，相似度均为97%。这两个OTU 的16S rDNA序列与已鉴定的最相近可培养产甲烷菌短柄产甲烷杆菌NT7的相似度也为97%。总之，在与从山羊瘤胃分离的厌氧真菌的共培养物中存在多种未鉴定的瘤胃产甲烷菌物种。
