T-RFLP分析厌氧真菌传代频率对共存产甲烷菌菌群的影响

Effects of transfer frequency on community of methanogens co-cultured with anaerobic fungi by T-RFLP

【目的】建立瘤胃产甲烷菌T-RFLP多样性分析方法,并研究厌氧真菌与产甲烷菌共培养液在不同时间传代对共存产甲烷菌菌群的影响。【方法】利用产甲烷菌mcrA基因特异性引物PCR扩增后,选择合适内切酶对扩增产物进行内切,分析内切后末端片段长度多态性,测定共培养液在不同传代频率时共存产甲烷菌多样性的变化。【结果】利用MspI内切酶分析发现,末端片段长度约为470bp的产甲烷菌为共培养液中的优势甲烷菌,共培养液传代至第15代时,片段长度约为130bp和200bp的产甲烷菌也成为共培养中的优势菌株;比较发现,TaqI能更好地内切共培养液中甲烷菌mcrA基因序列,瘤胃内容物及3d传代共培养液中产甲烷菌主要为末端片段长度约为70、100、200、270、300、330和470bp的菌株,共培养液在体外传代培养过程中,末端片段长度约为70、100、270和470bp的产甲烷菌变化更为显著。TaqI比较分析不同传代频率(3、5和7d)对共培养液中产甲烷菌菌群结构的影响表明,3d传代的共培养液中产甲烷菌菌群与瘤胃内容物较为相似,而5d和7d传代的共培养液中产甲烷菌菌群间差异较小,但与瘤胃内容物差异较大,导致不同传代频率的共培养液中产甲烷菌菌群间显著差异的最主要菌株为末端片段长度约为100bp的产甲烷菌,其次为末端片段长度约为70bp和270bp的产甲烷菌。【结论】利用建立的快速可行的瘤胃产甲烷菌T-RFLP方法分析表明,传代频率显著影响厌氧真菌与产甲烷菌共培养液中产甲烷菌的菌群结构,3d传代共培养液内产甲烷菌菌群与瘤胃内容物更相似。

[Objective] A molecular-based approach,terminal restriction fragment length polymorphism（T-RFLP）,for community analysis of ruminal methanogens was developed and used to assess the effects of transfer frequency on the community of methanogens co-cultured with anaerobic fungi.[Methods] The specific primers for mcr A genes were used to amplify the mcr A genes of methanogens and the amplicons were then digested with restriction enzymes.The size of each of the individual resulting terminal fragments were detected using DNA sequencer.[Results] With the analysis by Msp I,the dominant methanogens co-cultured with anaerobic fungi were those with terminal fragment of 470 bp and those of 130 bp and 200 bp were also dominant in the 15 th transfers of 3-day co-cultures.Comparative study with Taq I showed that the dominant methanogens both in rumen digesta and 3-day co-cultures were those with terminal fragment of 70,100,200,270,300,330 and 470 bp.The methanogens of 70,100,270 and 470 bp changed dramatically during in vitro transfers.Subsequently,the effects of transfer frequency on community of methanogens co-cultured with anaerobic fungi were assessed by Taq I and results showed that the community of methanogens in 3-day co-cultures was similar with rumen digesta,while they are significantly different from those in 5- and 7-day co-cultures,which was resulted from the shift of methanogens with terminal length of 100,70 and 270 bp.[Conclusion] The molecular-based approach,T-RFLP,was suitable for analysis of ruminal methanogens.The community of methanogens co-cultured with anaerobic fungi was significantly affected by transfer frequency of the co-cultures,and that in 3-day co-cultures was similar to that in the rumen digesta.