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堆肥环境中高浓度腐殖酸的存在阻碍了对这个环境中的未培养微生物的宏基因组研究。我们提出了一个确实可行的提取堆肥环境DNA的方法,这个方法通过使用Sephadex G200+酸洗PVPP层析柱与电洗脱两步纯化的方法成功地纯化堆肥环境来源的DNA,用提取的DNA成功构建了一个包含约10万个克隆的柯斯质粒文库。从这个文库中筛选到一个新的β-葡萄糖苷酶基因。针对文库低的阳性筛选率问题,利用分子技术研究了不同的分离速度对提取到的总DNA中真核生物DNA量的影响,以减少文库中真核生物DNA的污染。
The presence of high concentrations of humic acid in the composting environment hinders the metagenomic study of uncultured microorganisms in this environment. We propose a viable method of extracting DNA from composting environments by successfully purifying composting environment-derived DNA using a Sephadex G200 + acid-washed PVPP column and electro-elution two-step purification, and successfully constructed with the extracted DNA A cosmids library containing about 100,000 clones. A new β-glucosidase gene was screened from this library. In view of the low positive screening rate of the library, molecular techniques were used to study the effect of different separation rates on the amount of eukaryotic DNA extracted from the total DNA so as to reduce the contamination of eukaryotic DNA in the library.