堆肥宏基因组柯斯质粒文库的构建和DNA提取技术的改进

Construction of Cosmid Library of Metagenome Derived from Compost Sample and the Improvement of DNA Isolation Techniques

摘要:

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, 用这个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。

关键词:堆肥, 宏基因组, DNA提取纯化, 柯斯质粒文库

Abstract:

Abundant humic acid present in compost hinder the metagenomic study of uncultured microorganisms in compost environment. We developed an effective method for isolating metagenomic DNA from compost samples. By combinations of Sephadex G200 + acid washed PVPP column and electroelution techniques, this method was effective in purifying of metagenomic DNA from compost samples. With this purified DNA, we succeeded in constructing a cosmid library containing about one hundred thousands clones. And a novel β-glucosidase gene was cloned and sequenced from this library. Targeting the problem of low frequency of positive clones in the library, we also studied the effects of different centrifugal speeds on the content of contaminated eukaryotic DNA in the isolated metagenomic DNA, in order to preclude the presence of eukaryotic DNA in metagenomic library.

Key words:Compost, Metagenome, DNA isolation and purification, Cosmid library