沈阳农业大学学报2017，Vol.48Issue(4)：467-471,5.DOI:10.3969/j.issn.1000-1700.2017.04.013
添加生物炭土壤中总DNA最佳提取方法的筛选
Selection of Different DNA Extraction Methods for Soil Treated with Biochar
摘要
Abstract
Isolation of total microbial DNA with high purity is the basis for soil metagenomic study, and it is also the key to analyze the influence of biochar on soil microbial community structure. Because of the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is more difficult than the extraction of DNA from other environmental samples. In order to find a suitable method to extract DNA from biochar-amended soil, 5 different methods were used, including E.Z.N.A. Soil DNA Kit method (method 1), modified E.Z.N. A. Soil DNA Kit method (method 2), SDS-glass beads method (method 3), Mo-Bio Power Soil DNA Isolation Kit method (method 4) and modified Mo-Bio Power Soil DNA Isolation Kit method (method 5). The extracted DNA was subjected to agarose gel electrophoresis analysis and its concentration and purity were determined. The results showed that all of the 5 methods could obtain the soil DNA. The size of DNA was uniform, and the band was relatively simple. At the same time, the concentrations of DNA extracted from the two mixed soil samples were from 3.22 μg·g-1 dry soil to 37.80 μg·g-1 dry soil. The DNA extraction of soil sample 1 was significantly larger than that of soil sample 2. However, there were significant differences in the yield and quality of DNA extracted by different methods. Among them, the DNA yield of method 3 was the highest, the two soil samples were 37.80 μg·g-1 dry soil and 10.78 μg·g-1 dry soil respectively. But its DNA purity was the lowest. The A260/A230 values of the two soil samples were only 0.664 and 0.684, and the A260/A280 values were also less than 1.6, indicating that there was serious contamination of protein and humic acid in the extracted products and they could not be directly used for PCR amplification. Although the DNA yield of method 2 was relatively lower, its DNA purity was the highest. The A260/A280 values of the two soil samples were 1.854 and 1.844, respectively, and the A260/A230 values were 1.857 and 1.663, respectively, which indicated that the removal of protein and humic acid was more thorough. Because method 2 was improved on the basis of a commercial kit, it was simple, time-saving, and reproducible. Based on the results obtained in the study, the modified E.Z.N.A. Soil DNA Kit method could obtain the highest quantity of soil DNA, and it is an ideal method for extracting DNA from biochar-amended soil to meet the requirement of constructing metagenomic library.关键词
生物炭/土壤微生物/DNA提取/宏基因组Key words
biochar/soil microorganism/DNA extraction/metagenome分类
农业科技引用本文复制引用
孟庆阳,唐文竹..添加生物炭土壤中总DNA最佳提取方法的筛选[J].沈阳农业大学学报,2017,48(4):467-471,5.基金项目
公益性行业(农业)科研专项项目(201303095) （农业）
