野生秦岭羚牛瘤胃产甲烷古菌多样性研究

目的]了解野生秦岭羚牛瘤胃产甲烷古菌的组成及其多样性。方法]提取野生秦岭羚牛瘤胃内容物总DNA,选用产甲烷菌特异性引物Met86F和Met1340R,对总DNA进行16S rRNA特异性扩增,构建秦岭羚牛瘤胃产甲烷古菌16S rRNA克隆文库,对阳性克隆进行PCR-RFLP(限制性内切酶片段长度多态性)分析,并对HaeⅢ酶切带谱不同的菌液进行测序,构建系统发育树。结果]根据酶切带谱分析和测序结果的不同,将随机挑取的105个阳性克隆归为14个不同的可操作分类单元(OTUs)。系统发育分析表明,这些克隆序列均位于广域古菌门(Euryarchaeota),隶属甲烷杆菌科(Methanobacteriaceae,65.71%)、Methanomassiliicoccus(29.52%)和Methanosarcinaceae(4.76%)3个科。其中有57个序列与可培养菌相似度在97%以上,分属于Methanobrevibacter millerae、Methanobrevibacter olleyae和Methanobrevibacter thaueri。其余48个序列与未培养菌相似度较高,占11个OTUs。结论]野生秦岭羚牛瘤胃产甲烷古菌多样性比较丰富,且可能存在新的分类单元。

Study on the Diversity of Methanogenic Archaea in the Rumen of Wild Budorcas taxicolor bedfordi

Objective]To investigate the composition and diversity of methanogenic archaea in the rumen of wild Budorcas taxicolor bedfordi. Method]Total DNA was extracted from rumen content of wild B.taxicolor.A pair of methanogenic archaea's specific PCR primers were used for 16S rRNA specific amplification and a clone library was constructed for the DNA samples.Positive clones were analyzed by PCR-restriction frag-ment length polymorphism(PCR-RFLP)and the clones with unique HaeⅢpatterns were selected for sequencing and constructing 16S rRNA gene phylogenetic tree.Result]Based on HaeⅢdigestion and sequencing results,a total of 105 positive clones randomly screened from the library were classified into 14 operational taxonomic units(OTUs).Phylogenetic analysis showed that these clone squences belonged to Euryarchaeota and they were divided into three families:Methanobacteriaceae,Methanomassiliicoccus and Methanosarcinaceae.The similarity between 57 se-quences and cultured archaea was above 97%and they belonged to Methanobrevibacter millerae,Methanobrevibacter olleyae and Methanobrevibacter thaueri.Other 48 sequences had an higher similarity with uncultured archae,containing 11 OTUs.Conclusion]The methanogenic archaea in the rumen of wild B.taxicolor had an abundant diversity methanogens and there might be a new taxon.