Amplification of uncultured single-stranded DNA viruses from rice paddy soil
Amplification of uncultured single-stranded DNA viruses from rice paddy soil
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DOI：
10.1128/aem.01275-08
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发表时间：
2008-10-01
影响因子：
4.4
通讯作者：
Bae, Jin-Woo
中科院分区：
文献类型：
--
作者：
Kim, Kyoung-Ho;Chang, Ho-Won;Bae, Jin-Woo
病毒是土壤中数量最多的生物实体；然而，人们对它们在这种环境中的多样性知之甚少。为了探索土壤病毒的遗传多样性，在进行宏基因组研究之前，我们采用离心分离和顺序过滤的方法分离病毒。采用多位移扩增（MDA）技术，利用phi 29聚合酶和随机六聚体进行等温全基因组扩增，扩增病毒DNA并构建克隆文库进行宏基因组测序。通过MDA方法可以同时研究单链DNA （ssDNA）病毒和双链DNA病毒的多样性。相反，通过消除MDA反应中的变性步骤，只能选择性地探索ssDNA病毒多样性。无论变性步骤如何，超过60%的土壤宏基因组序列与先前报道的病毒序列没有显着匹配（e值标准，0.001）。那些被认为是重要的命中也与已知的ssDNA病毒有远亲关系（平均氨基酸相似性，约为34%）。系统发育分析表明，从宏基因组序列中获得的与ssDNA病毒相关的复制相关蛋白（最常检测到的蛋白）具有多样性和新颖性。假定的与已知病毒无关的ssDNA病毒的环状基因组成分由宏基因组序列组装而成。总之，土壤中ssDNA病毒的多样性比以前认为的要复杂得多。因此，土壤是以前未知的ssDNA病毒的丰富宝库。
Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with phi 29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.