富集培养及高质量DNA提取有利于从土壤宏基因组中获取新卤醇脱卤酶基因

Enrichment Cultivation and High Quality DNA Extraction Represent an Available Strategy for Isolation New Halohydrin Dehalogenase Genes from Soil Metagenome

目的:宏基因组技术作为一种不依赖于微生物纯培养的新方法,在挖掘新基因方面具有极大的潜力。本研究旨在建立一种从土壤中高效获取卤醇脱卤酶新基因的策略。方法:通过对现有DNA提取方法进行改进,同时结合富集培养途径以提高土壤宏基因组DNA质量和特异性;在此基础上,应用T-Coffee及CDEHOP程序设计特异引物并对目的基因进行扩增,同时采用正交法设计优化扩增条件,以提高获得卤醇脱卤酶基因的效率。结果:应用改进法提取的DNA质量较改进前有大幅度提高,其D260nm/D280nm及D260nm/D230nm值均大于1.8,且可以不经纯化直接用于PCR和相关酶切实验;PCR扩增目标基因的特异性增强,其中用经富集培养后所得DNA为模板扩增目标基因的特异性最强,TA克隆测序阳性结果比例最高。结论:富集培养和高质量DNA的获得有助于基于宏基因组途径获取新基因。

Objective： Metagenomics,being a novel culture-independent genomic analysis technique,has potentials to recover functional genes from environmental resource.The study aims at the development of a methodology that relates to efficient isolation of new halohydrin dehalogenase genes from soil.Methods： Such strategies are usually initiated by the extraction of metagenomic DNA,thus,an improved method for the isolation of high-quality metagenomic DNA from soil was first established.In addition,to enhance gene specificity of the isolated DNA an enrichment cultivation approach was applied.A sequence-based screening strategy was used to isolate colonies that may contain new halohydrin dehalogenase genes.For this,primers were designed by using T-Coffee and CDEHOP program and PCR conditions were optimized using an orthogonal array method.Results： The isolated DNA can be directed applied to PCR amplification using Tag polymerase and restriction enzyme digestion systems without the needs of purification.The probability of isolation new halohydrin dehalogenase genes using sequence-based screen-ing strategy was greatly enhanced by applying an enrichment cultivation approach.Conclusion： The strategy,which comprised an improved DNA extraction methodology,enrichment cultivation and sequence-based screening technologies,sets a solid base for isolation of new functional genes from soil.