变性梯度凝胶电泳(DGGE)在微生物生态学中的应用

Application of denaturing gradient gel electrophoresis (DGGE) in microbial ecology

由于从环境样品中分离和培养细菌的困难 ,分子生物学方法已发展用来描述和鉴定微生物群落。近年来基于 DNA方法的群落分析得到了迅速的发展 ,如 PCR扩增技术 ,克隆文库法 ,荧光原位杂交法 ,限制性酶切片段长度多态性法 ,变性和温度梯度凝胶电泳法。DGGE已广泛用于分析自然环境中细菌、蓝细菌 ,古菌、微微型真核生物、真核生物和病毒群落的生物多样性。这一技术能够提供群落中优势种类信息和同时分析多个样品。具有可重复和容易操作等特点 ,适合于调查种群的时空变化 ,并且可通过对切下的带进行序列分析或与特异性探针杂交分析鉴定群落成员。DGGE分析微生物群落的一般步骤如下 :一是核酸的提取 ,二是 1 6Sr RNA,1 8S r RNA或功能基因如可容性甲烷加单氧酶羟化酶基因 ( mmo X)和氨加单氧酶 α-亚单位基因 ( amo A)片段的扩增 ,三是通过DGGE分析 PCR产物。DGGE使用具有化学变性剂梯度的聚丙烯酰胺凝胶 ,该凝胶能够有区别的解链PCR扩增产物。由 PCR产生的不同的 DNA片段长度相同但核苷酸序列不同。因此不同的双链 DNA片段由于沿着化学梯度的不同解链行为将在凝胶的不同位置上停止迁移 [5] 。 DNA解链行为的不同导致一个凝胶带图案 ,该图案是微生物群落中主要种类的一个轮廓。 DGGE使用所有生物中保守的基因?

Because of the difficulty associated with isolating and culturing bacteria from environmental samples, alternative methods based on molecular techniques have been developed to describe and identify microbial communities. Recent years have witnessed a rapid development of DNA-based methods for community analysis such as PCR amplification, clone libraries, fluorescent %in-situ% hybridisation, restriction fragment length polymorphism, denaturing and temperature gradient gel electrophoresis. ;DGGE has been widely used in analyzing the biodiversity of bacterial, cyanobacterial, archaeal, picoeukaryotic, eukaryotic and viral communities in natural habitats. This technique can provide information on the predominant species in a community and analyze multiple samples simultaneously. The reproducibility and ease-of use of this technique permit investigation of the spatial and temporal variability of the population and identification of community members by sequencing of excised bands or by hybridization analysis with specific probes.;The general procedure for DGGE analysis of communities of microorganisms is as follows: First, nucleic acid extraction; Second, amplification of genes encoding the 16S rRNA, 18S rRNA or functional genes such as mmoX, amoA; Third, analyze PCR products by DGGE. DGGE employs a polyacrylamide gel with a chemical denaturing gradient that differentially melts the PCR amplified products. The different DNA fragments generated by PCR are of the same length but differ in the nucleotide sequence. Therefore, the different double stranded DNA fragments will stop migrating at different position in the DGGE gel due to their different melting behavior along the chemical gradient. The differences in the melting behavior of the DNA lead to a banding pattern, which is a profile of the predominant species present in the community. DGGE utilizes a gene fragment conserved among all organisms for example 16S rRNA gene fragment for bacteria and 18S rRNA for fungi.;It must be emphasized that, as with other molecular methods, DGGE is not free of biases. One of these biases is that DGGE allows separation only of small fragments, which limits the amount of sequence information for phylogenetic comparison as well as probe design. In some circumstances, identification of community structure to the species level is problematic due to multiple copies of the gene of interest resulting in more than one band in a single species. In addition, this technique carries the inherent problem of heterogeneity between copies of, for example, the 16S rDNA in a single bacterial species, which leads to an overestimation of the number of microbes within natural communities.;DGGE is a powerful tool for the analysis of microbial communities. However, in order to reduce potential biases and limitations of DGGE and other techniques, it is suggested that researchers combine DGGE with other molecular and microbiological techniques to obtain a more detailed view of microbial community structure and function.