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PCR-DGGE技术在农田土壤微生物多样性研究中的应用
引用本文:罗海峰,齐鸿雁,薛凯,张洪勋.PCR-DGGE技术在农田土壤微生物多样性研究中的应用[J].生态学报,2003,23(8):1570-1575.
作者姓名:罗海峰  齐鸿雁  薛凯  张洪勋
作者单位:中国科学院生态环境研究中心环境生物技术研究室,北京,100085
基金项目:国家“十五”科技攻关重点项目 ( 2 0 0 1 BA90 3 B)~~
摘    要:变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。

关 键 词:变性梯度凝胶电泳  DGGE  基因组DNA  16S  rRNA  微生物多样性  农田土壤
文章编号:1000-0933(2003)08-1570-06
收稿时间:1/3/2003 12:00:00 AM
修稿时间:4/1/2003 12:00:00 AM

A preliminary application of PCR-DGGE to study microbial diversity in soil
LUO Haifeng,QI Hongyan,XUE Kai and ZHANG Hongxun.A preliminary application of PCR-DGGE to study microbial diversity in soil[J].Acta Ecologica Sinica,2003,23(8):1570-1575.
Authors:LUO Haifeng  QI Hongyan  XUE Kai and ZHANG Hongxun
Institution:Environmental Biotechnology Lab.; Research Center for Eco-environmental Sciences; Chinese Academy of Sciences; Beijing; China
Abstract:Few traditional methods are applicable in study of the microbial community in the field of microbial ecology in recent years, since most of them rely on the microscope and cultivation of bacteria on laboratory media, and it had been proved that only about 1% of total number of actively respiring bacterial cells can be cultured on laboratory media. In addition, less than 1% bacterial species have been isolated and characterized from soil samples. As a new DNA fingerprinting technique, denaturing gradient gel electrophoresis (DGGE) can be used to analyze the microbial diversity in different environmental samples. Application of DGGE in this study had provided new ideas for study of the microbial community in soil. Seven soils in different farmland were sampled and their genomic DNA of microbial community were extracted directly in chemical method, which is based on lysis with a high-salt extraction buffer (1^5mol/L NaCl) and extend heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), CTAB and proteinase K. For seven soils with different texture in this study, different DNA yields were obtained. After purification of the genomic DNA by DNA gel Recovery kit, the 16S rRNA genes (V3 region) were amplified by using the specific primers (F 357GC and R 518). The result of agarose gel (1.7%) electrophoresis showed that the PCR products were about 230 bp. These amplified DNA fragments were then separated by paralleled DGGE with the denaturant (urea and formamide) from 30% to 50%. The profile of DGGE showed that different soils had the different bands' patterns. DGGE could clearly separate the amplified fragments of 16S rRNA genes (V3 region) of different microbial community in soils, which make it possible to identify and define these 16S rDNA fragments by sequencing. For all DGGE profiles, there exist some common bands in all soil samples, which indicated that some kinds of microorganisms exist in each soil. On the other hand, the specific bands in some soils showed that different soils had the their own specific microorganisms. Compared to the traditional methods, DGGE could give more comprehensive information about the microbial community in soils. Our results showed that DGGE is a new and effective technique in analyzing microbial diversity.
Keywords:denaturing gradient gel electrophoresis (DGGE)  genomic DNA  16S ribosome RNA  microbial diversity  
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