不同无土栽培基质微生物DNA提取方法研究

Study on Extraction Methods for Total Microorganism DNA from Soilless Substrate

用4种DNA提取方法对3种栽培基质中微生物的DNA浓度、腐殖酸去除率、16S rDNA的PCR扩增、扩增片段限制性内切酶和变性梯度凝胶电泳结果进行比较。结果表明:采用含CaCO3的缓冲液预洗,用CTAB缓冲液和蛋白质酶K共同作用以裂解细胞,同时添加2%CaCl2可有效去除腐殖酸;用PEG 8000沉淀DNA和Sephadex G-200纯化,可提高DNA质量;所得DNA适用于PCR扩增及ARDRA技术的限制酶切分析;在DGGE分析中此方法更能显示微生物种群的多样性。

A comparative study was conducted on the results of DNA concentration, removal rate of humic acids, PCR amplification of 16S rDNA, restriction endonucleases of amplified fragment and the denaturing gradient gel electrophoresis of the microorganisms isolated from 3 types culture substrates with 4 DNA extraction protocols. The results showed that the humic acids could be effectively removed by precleaning the samples with CaCO3 buff- er, cell lysis process synergically assisted with CTAB buffer and proteinase K and adding 2% CaClfinto the reaction solution. The DNA quality was improved after being precipitated by PEG 8000 and then purified by Sephadex G -200 spin column. The DNA obtained by this way was qualified for PCR amplification and restriction endonucle- ases analysis with ARDRA technique. This kind of DNA extraction might be applied to DGGE analyses and to ex- press the diversity of microbial populations.