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  

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

作　　者：成艳芬[1] 朱伟云[1] 

机构地区：[1]南京农业大学消化道微生物研究室,南京210095

出　　处：《微生物学报》2009年第4期504-511,共8页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30530560)~~

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、Neocalli mastix属菌株(YC501与YC502)的ARISA分析结果显示:菌株YC301得到一个长度为389.67bp的片段;YC501得到4个长度分别为415.91、425.69、437.31和438.46bp的片段;YC502所得片段与YC501相似。ARISA分析来自瘤胃内容物的厌氧真菌与产甲烷菌共培养液中厌氧真菌多样性显示:共培养液在体外传代培养过程中,厌氧真菌多样性降低,ARISA扩增片段长度为354～375及425～438bp的厌氧真菌在体外传代中消失,片段长度为383、389～391及413～418bp的厌氧真菌是体外共培养液中的优势菌,在传代过程中稳定存在。对厌氧真菌与产甲烷菌共培养液及去除产甲烷菌的真菌培养液研究表明:产甲烷菌影响培养液中真菌的种类,当产甲烷菌存在时,共培养液中主要是片段长度为383.51、391.44和413.55bp的厌氧真菌;而添加氯霉素去除甲烷菌后,培养液中主要是片段长度为415.80、425.66、437.46和438.47bp的厌氧真菌。【结论】本文建立了快速可行的厌氧真菌多样性分析方法。ARISA分析表明厌氧真菌与产甲烷菌共培养液在传代过程中真菌多样性降低,共培养液传代4次后,厌氧真菌菌群趋于稳定。去除产甲烷菌后,共培养液中厌氧真菌的菌群结构发生改变。[Objective] A molecular-based approach for anaerobic funga： 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 subcuhured 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 subcuhured 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-cuhuers. The anaerobic fungi represented by 354-375 and 425-438 bp in the amplified ribosomal intergenic spacer analysis profiles were not deteched in the co-cultures after the second subcultures and the anaerobic fungi represented by 383, 389-391 and 413-418 bp were dominant along with 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 4^th subculture of the co-cuhures. The anaerobic fungal community was different in

关 键 词：厌氧真菌 产甲烷菌 ARISA 

分 类 号：S852.6[农业科学—基础兽医学]