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作 者 ：赵志祥,罗坤,陈国华,杨宇红,茆振川,刘二明*,谢丙炎*

期 刊 ：生态学报 2010年 30卷 14期 页码：3849~3857

关键词：土壤细菌多样性；末端测序；16S rRNA基因克隆文库；宏基因组文库；

Keywords:soil bacterial diversity, end-random sequencing, 16S rRNA gene library, metagenomic library,

摘 要 ：土壤细菌在温室土壤环境中具有十分重要的生态功能，与温室作物以及微生物内部存在互作关系。研究土壤细菌的群落结构组成，有助于了解土地利用变化与生态环境效应之间的关系。结合16S rRNA基因克隆文库和宏基因组末端测序对温室黄瓜根围土壤细菌的多样性进行了分析。在16S文库中，根据97%的序列相似性水平划分OTU，共有35个OTU，其中优势菌群是γ-Proteobacteria，其次为Firmicutes，Bacillus为优势细菌。在纲分类水平上，16S文库和宏基因组末端测序结果均包含γ-Proteobacteria、α-Proteobacteria、δ-Proteobacteria、β-Proteobacteria、Actinomycetales和Firmicutes，各纲比例有差别；在优势种群属水平上，末端测序的结果包含的属多于16S文库（40>35）；在优势细菌种类上，两者反映的结果一致，均为Bacillus。但是，宏基因组末端测序包含了大多数的弱势种群，更能反映细菌多样性的真实水平。与露地土壤细菌16S文库相比较，土壤细菌多样性降低，这可能与温室多年连作，种植蔬菜种类单一直接相关。

Abstract:Soil bacteria play an important ecological role in the ecosystem of soil in greenhouse and in the exchange of energy and substance, and have a significant inter- or intra interaction between bacterial community and greenhouse crops. Understanding of the composition of soil bacterial communities is benefit to reveal the relationship between the change of land use and the effects of entironment. In this study, total microbial genomic DNA from soil was extracted and purified by density gradient centrifugation, and 16S rRNA gene was amplified using universal primer, 27F and 1492R, and the product of PCR was recycled, ligated, transformed and screened using blue-white selection, and then was constructed 16S rRNA gene clone library. Partial total DNA from soil was treated with end repairing, ligated to Fosmid vector, PCC2FOS, packed using lambda packaging extracts, transfected with E.coli EPI300, and then was constructed metagenomic Fosmid library. The result showed that the 16S rRNA gene clone library was dominated by bacteria belonged to γ-Proteobacteria, which followed by Firmicutes, especially Bacillus, which constituted 32.2% of 16S rRNA gene clone library. The groups including α-Proteobacteria,δ-Proteobacteria and Actinomycetales were minority in the 16S rRNA gene clone library. In addition, 35 OTUs were obtained from the 16S rRNA gene clone library according to sequences similarity of 97%. From the result of end sequencing from metagenomic library, γ-Proteobacteria and Firmicutes were also dominant groups, which constituted 26.1% and 19.8%, respectively. At the level of class, the results from 16S rRNA gene clone library and metagenomic library were also included γ-Proteobacteria,α-Proteobacteria,δ-Proteobacteria,β-Proteobacteria, Actinomycetales and Firmicutes, but the proportions of each class were various, especially Actinomycetales, which contained 2 clones from 16S rRNA gene clone library and constituted 0.7%, but contained 26 clones from metagenomic library and constituted 8.7%. In terms of predominant genus, the numbers of genus from end sequencing were higher than that of 16S rRNA gene clone library（40>35）. In term of dominant bacteria, the two methods both showed that Bacillus was predominant group. However, the result from end sequencing of clone from metagenome contained some minority of bacterial groups, which were not revealed by 16S rRNA gene clone library, such as Gemmatimonadetes, Chloroflexi, CFB group bacteria, Planctomycetes, Verrucomicrobia and Cyanobacteria. This discrepancy maybe reflected the substantial difference in two methods and probably results from the defects of 16S rRNA gene method, including PCR bias, chimeric gene, heteroduplex and mutation and so on. Comparing of bacterial community of the exposed soil, the bacterial diversity of soil in greenhouse was relatively low. This phenomenon was possibly directly caused by continuous cropping and single cropping.

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