适用变性梯度凝胶电泳分析的石油污染土壤微生物DNA模板制备的研究

DNA Preparation for Analyzing Petroleum Contaminated Soil Microorganism by Denaturing Gradient Gel Electrophoresis(DGGE)

[目的]为了快速、简便和经济地获得理化性质不同的石油污染土壤中微生物总DNA,以用于后续微生物群落结构分析及动态监测。[方法]采用3种提取方法对石油污染土壤中细菌总DNA进行提取,并通过DNA产量、纯度、片段大小、基因组完整性等对提取得到的DNA质量进行评价;并对16S rDNAV3可变区的PCR扩增产物进行DGGE分析。[结果]采用3种方法均能从石油污染土壤中提取得到相应的细菌DNA片段,且针对同一种提取方法,土壤理化性质的差异对DNA提取效果影响不明显,但不同方法提取得到的DNA在浓度和纯度上存在明显差异。采用3种方法提取得到的DNA量分别可达98.3、79.9和43.8 ng/μl。[结论]选取方法1提取基因组DNA并进一步纯化,纯化后的DNA分别采用引物F27/R1492和F357/R518进行16S rDNA及116S rDNAV3可变区的扩增,获得了条带清晰、浓度高、无污染的DNA目的片段;对其PCR扩增产物进行DGGE分析,得到的DGGE图谱可直观反应石油污染土壤中微生物的多样性及优势种群。

[Objective]In order to obtain total DNA in a rapid high-efficient and economic way from microorganism in petroleum contaminated soil with different properties.The total DNA can be used in the microbial community structural analysis and dynamic monitoring.[Methods] Three DNA extraction methods were used for isolation of total DNA form petroleum contaminated soil and the evaluation of DNA quality was based on the DNA yield,purity,fragment size and genome integrity.The amplification of 16S rDNA V3 variable region and the subsequent analysis of amplified products by DGGE.[Result]The results showed that three methods can extract corresponding DNA fragment from samples,and for the same extraction method,different properties soil had no obvious effect on the DNA extraction;but different methods had obvious distinction in the concentration and purity of extracted DNA.The extracted DNA yield of three methods are up to 98.3,79.9 and 48.3 ng/μl,respectively.[Conclusion]So the genomic DNA was extracted and further purified by the first method,the 16S rDNA and 16S rDNA V3 were amplified with universal primes F27/R1492 and F357/R518.The amplified DNA fragment were separated by parallel DGGE.The DGGE profiles can directly reflect the petroleum contaminated soil microbial diversity and dominant species.