Widmer Franco, Hartmann Martin, Frey Beat, Kölliker Roland
Molecular Ecology, Agroscope FAL Reckenholz, Swiss Federal Research Station for Agroecology and Agriculture, Zurich, Switzerland.
J Microbiol Methods. 2006 Sep;66(3):512-20. doi: 10.1016/j.mimet.2006.02.007. Epub 2006 Mar 6.
Cultivation-independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. Semi-automated terminal restriction fragment length polymorphism (T-RFLP) analyses yield high-resolution genetic profiles of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring. A serious limitation of T-RFLP analyses has been the inability to reliably affiliate observed terminal restriction fragments (T-RF) to phylogenetic groups. In the study presented here, we were able to overcome this limitation of T-RFLP. With a combination of adapter ligation, fragment size selection, and re-amplification with adapter site specific PCR, we were able to isolate a T-RF-fraction of a narrow size-range containing a T-RF that was significantly more abundant in heavy metal amended soils. Cloning the size-selected T-RF fraction allowed for the efficient isolation of clones containing this specific T-RF. Sequence determination and phylogenetic inference in RDP-II affiliated the sequence to unclassified cyanobacteria. Specific primer design and PCR amplification from bulk soil DNA allowed for independent confirmation of the results from bacterial T-RFLP and T-RF cloning. Our results show that specific T-RFs can be efficiently isolated and identified, and that the adapter ligation approach holds great potential for genetic profiling and for identification of community components of interest.
不依赖培养的土壤微生物群落结构分析经常被用于描述土壤微生物特征。半自动末端限制性片段长度多态性（T-RFLP）分析能够产生高度多样化土壤微生物群落的高分辨率遗传图谱，在土壤质量常规监测中具有很大的应用潜力。T-RFLP分析的一个严重局限是无法将观察到的末端限制性片段（T-RF）可靠地与系统发育类群联系起来。在本文介绍的研究中，我们能够克服T-RFLP的这一局限。通过连接接头、片段大小选择以及用接头位点特异性PCR重新扩增相结合的方法，我们能够分离出一个窄大小范围的T-RF组分，其中包含一个在重金属改良土壤中显著更丰富的T-RF。对大小选择后的T-RF组分进行克隆能够有效分离出包含该特定T-RF的克隆。在RDP-II中进行序列测定和系统发育推断，将该序列归为未分类的蓝细菌。从大量土壤DNA中设计特异性引物并进行PCR扩增，能够独立证实细菌T-RFLP和T-RF克隆的结果。我们的结果表明，特定的T-RF能够被有效分离和鉴定，并且接头连接方法在遗传图谱分析和感兴趣的群落组分鉴定方面具有很大潜力。
