Assessment of bacterial community structure in soil by polymerase chain reaction and denaturing gradient gel electrophoresis.
Assessment of bacterial community structure in soil by polymerase chain reaction and denaturing gradient gel electrophoresis.
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DOI：
10.1016/s0167-7012(99)00054-8
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发表时间：
1999-10-01
影响因子：
2.2
通讯作者：
van Elsas, JD
中科院分区：
文献类型：
--
作者：
Gelsomino, A;Keijzer-Wolters, AC;van Elsas, JD
采用DNA提取和分子指纹分析方法，研究了Flevo粉壤土（FSL）和其他15种土壤中细菌群落结构。用直接法提取和纯化了土壤总DNA，得到了所有土壤的高分子量DNA。然后用细菌引物通过PCR扩增16 S rRNA基因的可变区，得到通过变性梯度凝胶电泳（DGGE）可分离的扩增子的混合物。FSL土壤的DGGE图谱表明了土壤细菌的优势类型，这一点通过评估阴沟肠杆菌和节杆菌的扩增来证明。当这些靶标以每克干土约5 × 10（6）基因组当量存在于土壤DNA中时，几乎检测不到条带，并且在高27倍的水平下产生强条带。FSL土壤的PCR-DGGE分析具有高度的重复性。此外，不同的单一与复合表土样品产生了类似的DGGE配置文件的主要波段。此外，沿沿着垂直土芯（0-45厘米深）取样显示DGGE剖面相对稳定。从不同团聚体大小的分数，这种土壤中获得的DNA产生的配置文件也是类似的主带。此外，FSL表土样品超过1年的时间（休闲土壤）产生稳定的配置文件。这些数据表明，由此确定的土壤细菌群落占主导地位的数量有限的稳定和普遍存在的类型。16个土壤，代表不同的类型和地理位置，进行了评估，其细菌DGGE配置文件的差异。有显着的差异，这些土壤中获得的配置文件。DGGE图谱显示，相似的土壤类型往往包含相似的优势细菌类型结构，这一假设得到了证实。(C)1999 Elsevier Science B. V.保留所有权利。
Bacterial community structure was studied in a Flevo silt loam (FSL) soil microplot, as well as in 15 other soils, by using DNA extraction followed by molecular fingerprinting. Total community DNA was extracted and purified by a direct method, which yielded amplifiable DNA of high molecular weight for all soils. A variable region of the 16S rRNA gene was then amplified by PCR with bacterial primers, resulting in a mixture of amplicons separable via denaturing gradient gel electrophoresis (DGGE). The DGGE profiles of FSL soil were indicative of dominant soil bacterial types, as evidenced by assessing the amplification of Enterobacter cloacae and Arthrobacter sp. targets in a soil DNA background. These targets produced barely detectable bands when present in soil DNA at roughly 5 X 10(6) genome equivalents per g dry soil, and strong bands at 27-fold higher levels. The PCR-DGGE analysis of the FSL soil was highly reproducible. Furthermore, different single versus composite topsoil samples yielded similar DGGE profiles with respect to major bands. In addition, samples taken along vertical soil cores (0-45 cm depth) revealed relative stability of the DGGE profiles. The profiles produced with DNA obtained from different aggregate size fractions of this soil were also similar with respect to the main bands. Moreover, FSL topsoil samples taken over a 1-year period (fallow soil) yielded stable profiles. These data suggested that the soil bacterial communities thus determined were dominated by a limited number of stable and ubiquitous types. The 16 soils, representing varying types and geographical locations, were assessed for differences in their bacterial DGGE profiles. There were striking differences between the profiles obtained for these soils. Evidence was found for the hypothesis that similar soil types tend to contain similar structures of the dominating bacterial types as revealed by the DGGE profiles. (C) 1999 Elsevier Science B.V. All rights reserved.