晋南牛瘤胃中古菌分子多样性的研究

Molecular diversity of rumen Archaea from Jinnan cattle

摘要:

采用3对古菌特异性引物扩增瘤胃古菌16S rRNA基因分别建立的克隆库来研究晋南牛瘤胃古菌的多样性。每个克隆库随机挑选100个克隆。引物Arch f364/1386建立的克隆库中, 克隆分为四类, 分别与四种甲烷短杆菌1Y (61%)、SM9 (23%)、NT7 (14%)和AK-87(2%)相似。引物1Af/1100Ar建立的克隆库中, 克隆分为两类, 分别与Methanobacterium aarhusense(72%) 和Methanosphaera stadtmanae DSM 3091 (28%)相似。引物Met86F/Met1340R建立的克隆库反映的古菌种类较为全面, 除以上四种甲烷短杆菌(所占比例分别为47%、26%、11%和3%)外, 还有Methanomicrobium mobile(2%)、以及类似Methanobacterium aarhusense(1%)和Methanosphaera stadtmanae(3%)的序列, 还有7%的未匹配序列。系统进化分析表明, 这些克隆属于Methanobrevibacter、Methanobacterium、Methanosphaera、Methanomicrobium, 和未知广域古菌等5个分支。有25类属于广域古菌的未知序列, 提示瘤胃中存在大量的未知产甲烷菌

Abstract:

Molecular diversities of rumen archaea of Jinnan (South Shanxi Province, China) cattle was analyzed and compared by 16S rRNA gene sequencing from three clone libraries generated using three different archaea-specific primer sets, respectively. DNA from rumen of 4 Jinnan cattle was extracted, and methanogen 16S rRNA gene was amplified using archaea-specific primer sets. Three clone libraries were generated by using vector pGEM-T and cloning into E.coli JM109. One hundred clones were randomly picked up for each library and RFLP was analyzed for each clone to obtain OTUs. Sequences from each OTU were analyzed and compared with available sequences in GenBank. The first library, generated with primers Arch f364/1386, produced four groups of sequences, affiliated with 4 Methanobrevibacter strains, 1Y (61% of clones), SM9 (23% of clones), NT7 (14% of clones), and AK-87(2% of clones). The second library, generated with primers 1 Af/1100Ar, two groups of sequences, one affiliated with Methanobacterium aarhusense(72% of clones) and the other with Methanosphaera stadtmanae DSM 3091 (28% of clones). The third library, generated with primers Met86F/Met1340R, produced a high degree of diversity. It included the sequence groups found in the first and the second libraries, as well as sequences affiliated with the Methanomicrobium mobile (2% of clones) and uncultured euryarchaeote sequences (7% of clones). The phylogenetic analysis indicated that archaea found in the three libraries were clustered in Methanobrevibacter, Methanobacterium, Methanosphaera, Methanomicrobium, and unidentified euryarchaeote of the Euryarcharota. There were 25 unidentified sequences belonged to Euryarchaeota. This suggests the existence of novel methanogens in the rumen of Jinnan cattle.