Schmalenberger Achim, Tebbe Christoph C
BITOK, Dr Hans-Frisch-Str 1-3, 95440 Bayreuth, Germany.
Mol Ecol. 2003 Jan;12(1):251-62. doi: 10.1046/j.1365-294x.2003.01716.x.
A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes and genetic profiling by single-strand conformation polymorphism (SSCP) was used to characterize the bacterial diversity inhabiting the rhizosphere of maize plants grown on an agricultural field. The community structures of two cultivars, a genetically engineered and a nonengineered variety, different herbicide regimes and soil tillage were compared with each other at two sampling dates. SSCP-profiles were generated with DNA from bacterial cell consortia with primers hybridizing to evolutionarily highly conserved rRNA gene regions. On silver-stained gels, each profile consisted of approx. 50 distinguishable bands. Similarity analyses of patterns recorded by digital image analyses could not detect any difference between cultivars or treatments that was greater than the variability between replicates. A total of 54 sequences recovered from different bands were identified and grouped into operational taxonomical units (OTUs). Surprisingly, only five of 40 OTUs contained sequences of both samplings. Three different bands from a profile were selected to test whether this small overlap was due to an incomplete recovery of sequences. From a faint band, two different OTUs were found when 12 clones were analysed, and from two strong bands 24 and 22 OTUs were detected from a total of 26 and 36 clones, respectively. The OTUs belonged to phylogenetically different groups of bacteria. Gene probes that were developed to target different bands of the profiles, however, indicated in Southern blot analyses that patterns between treatments, replicates and samplings, and even from two different growing seasons were highly conserved. Our study demonstrates that community profiles can consist of more sequences than detectable by staining and that gene probes in Southern blot can be a useful control to investigate the composition of microbial communities by genetic profiles.
一种基于聚合酶链反应（PCR）扩增的部分小亚基rRNA基因和单链构象多态性（SSCP）遗传图谱分析的非培养方法，用于表征生长在农田中的玉米植株根际的细菌多样性。在两个采样日期，对两个品种（一个转基因品种和一个非转基因品种）、不同除草剂处理方式和土壤耕作方式下的群落结构进行了相互比较。使用与进化上高度保守的rRNA基因区域杂交的引物，从细菌细胞联合体的DNA中生成SSCP图谱。在银染凝胶上，每个图谱大约由50条可区分的条带组成。通过数字图像分析记录的图谱相似性分析未检测到品种或处理之间存在大于重复样本间变异性的差异。从不同条带中回收的总共54个序列被鉴定并归类为操作分类单元（OTU）。令人惊讶的是，40个OTU中只有5个包含两个采样的序列。从一个图谱中选择了三个不同的条带，以测试这种小的重叠是否是由于序列回收不完全所致。对一个淡色条带分析12个克隆时发现了两个不同的OTU，对两个深色条带分别分析26个和36个克隆时，分别检测到24个和22个OTU。这些OTU属于系统发育上不同的细菌类群。然而，针对图谱不同条带开发的基因探针在Southern杂交分析中表明，处理、重复样本和采样之间，甚至来自两个不同生长季节的图谱模式都高度保守。我们的研究表明，群落图谱可能包含比染色可检测到的更多序列，并且Southern杂交中的基因探针可作为通过遗传图谱研究微生物群落组成的有用对照。
