Lovell Charles R, Decker Peter V, Bagwell Christopher E, Thompson Shelly, Matsui George Y
Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, United States.
J Microbiol Methods. 2008 May;73(2):160-71. doi: 10.1016/j.mimet.2008.02.005. Epub 2008 Feb 19.
Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and yielded 58 nonidentical nifH sequences from a total of 67 sequences determined. A clone library constructed using the GC-clamp nifH primers that were employed in the PCR-DGGE (designated the GC-Library) yielded 83 nonidentical sequences from a total of 257 nifH sequences. A second library constructed using an alternate set of nifH primers (N-Library) yielded 83 nonidentical sequences from a total of 138 nifH sequences. Rarefaction curves for the libraries did not reach saturation, although the GC-Library curve was substantially dampened and appeared to be closer to saturation than the N-Library curve. Phylogenetic analyses showed that DGGE gel band sequencing recovered nifH sequences that were frequently sampled in the GC-Library, as well as sequences that were infrequently sampled, and provided a species composition assessment that was robust, efficient, and relatively inexpensive to obtain. Further, the DGGE method permits a large number of samples to be examined for differences in banding patterns, after which bands of interest can be sampled for sequence determination.
对评估盐沼互花米草（Spartina alterniflora）根际固氮菌组合多样性的方法进行了研究。将nifH PCR-变性梯度凝胶电泳（DGGE）的有效性与nifH克隆文库分析的有效性进行了比较。对17条DGGE凝胶条带进行了测序，在总共测定的67条序列中产生了58条不同的nifH序列。使用PCR-DGGE中使用的GC夹nifH引物构建的克隆文库（称为GC文库），在总共257条nifH序列中产生了83条不同的序列。使用另一组nifH引物构建的第二个文库（N文库），在总共138条nifH序列中产生了83条不同的序列。文库的稀释曲线未达到饱和，尽管GC文库曲线明显变缓，并且似乎比N文库曲线更接近饱和。系统发育分析表明，DGGE凝胶条带测序回收了在GC文库中经常采样的nifH序列，以及很少采样的序列，并提供了一种可靠、高效且获取成本相对较低的物种组成评估。此外，DGGE方法允许对大量样品进行条带模式差异检查，之后可以对感兴趣的条带进行采样以进行序列测定。
