ARISA方法研究产甲烷菌共存及去除条件下瘤胃真菌多样性变化

摘要：【目的】建立厌氧真菌多样性分析方法，并研究厌氧真菌与产甲烷菌共培养液在传代过程中厌氧真菌的区系变化及共培养液中去除产甲烷菌条件下厌氧真菌多样性的变化。【方法】根据厌氧真菌ITS1序列长度多态性，设计厌氧真菌特异性引物，然后PCR扩增样品中厌氧真菌ITS1序列，在基因分析仪中分析PCR产物序列长度多态性，分析共培养液在传代过程中及共培养液中去除产甲烷菌后厌氧真菌多样性的变化。【结果】对瘤胃厌氧真菌Caecomyces属YC301菌株、Neocallimastix属菌株（YC501与YC502）的ARI

Diversity analysis of anaerobic fungi in the co-cultures with or without methanogens by amplified ribosomal intergenic spacer analysis

Abstract: Objective] A molecular-based approach for anaerobic fungal community analysis was developed. The diversity of anaerobic fungi in the co-cultures with or without methanogens was analyzed by amplified ribosomal intergenic spacer analysis. Methods] Co-cultures of anaerobic fungi and methanogens were obtained from rumen digesta using anaerobic fungal medium and the addition of penicillin and streptomycin and ampicillin alternatively and then subcultured 15 times by transferring cultures every 3 d separately for each replicate. At the end of the third subcultures, the co-cultures were inoculated to another bottles adding with chloramphenicol to obtain fungal cultures without methanogens. Total DNA from the original rumen digesta and subcultured co-cultures and fungal cultures was used for amplified ribosomal intergenic spacer analysis. Results] The diversity of anaerobic fungi decreased corresponding with the subculture of the co-cultuers. The anaerobic fungi represented by 354-375 and 425-438 bp in the amplified ribosomal intergenic spacer analysis profiles were missing in the co-cultures after the second subcultures and the anaerobic fungi represented by 383, 389-391 and 413-418 bp were dominant although the subcultures. The community of anaerobic fungi was different in the co-cultures with or without methanogens. The anaerobic fungi represented by 383.51, 391.44 and 413.55 bp in the amplified ribosomal intergenic spacer analysis profiles were dominant in the co-cultures with methanogens, while the anaerobic fungi represented by 415.80, 425.66, 437.46 and 438.47 bp were dominant in the co-cultures without methangens. Conclusion] The molecular-based approach amplified ribosomal intergenic spacer analysis was suitable for analysis of anaerobic fungi in the environmental samples. The diversity of anaerobic fungi decreased along with the subculture of the co-cultures and the anaerobic fungal community became stable after the 4th subculture of the co-cultures. The anaerobic fungal community was different in the co-cultures with or without methanogens.