利用土壤宏基因组技术定向筛选抗生素产生菌的方法学建立

A method of screening for antibiotic microorganism producing strains from soil by metagenomics

摘要:

为了快速从土壤中定向筛选抗生素产生菌, 本研究利用宏基因组技术进行了土壤中抗生素产生菌快速筛选方法的探索。对6 种不同土质的土壤利用液氮冷冻法提取土壤基因组DNA 并用PVPP柱层析法进行纯化, 每克湿土可得到32.25?61.25 μg DNA, 片段大小为23 kb左右, 说明宏基因组DNA样品完整, 16S rDNA的 PCR结果证明了该基因组DNA纯度可用于后期的分子操作。利用Herbimycin产生菌掺入法进行了抗生素产生菌筛选方法学的验证。结果证明, 每克湿土掺入103个Herbimycin产生菌时, 利用Herbimycin合成基因簇的特定引物即可从所提取的土壤基因组DNA中扩增出目标条带, 并且可以利用传统菌种分离方法从土壤中重新分离出来被掺入到土壤的Herbimycin产生菌。这些结果证明本研究建立的定向抗生素产生菌筛选方法, 具有快捷、灵敏的特点, 大大减少传统筛选方法的工作强度和时间, 为微生物新药的研发建立一种全新的研究方法。

Abstract:

A new method was developed to screen for antibiotic microorganism producing strains from soil using metagenomics. Six different environmental soils metagenomics DNA were extracted with liquid nitrogen freezing method and purified by polyvinylpolypyrrolidone (PVPP) column chromatography. Soil DNA of 32.25?61.25 μg with DNA length about 23.1 kb can be extracted from 1 g sample. 16S rDNA can be amplified from the purified soil DNA which proved that the extracted soil DNA is suitable for following experiments. In addition, the sensitivity and feasibility of this screening method is evaluated by addition of Herbimycin producing strain into a negative soil. The result showed that when the soil contains 103 CFU/g of Herbimycin producing strain, the Herbimycin biosynthesis gene can be detected by PCR and the incorporated known strains can also be isolated. These results suggest that soil metagenomics can be applied for screening certain antibiotic microorganism producing strains which is sensitive and rapid, and is more efficient than the traditional methods.