西藏米拉山高寒草甸土壤微生物DNA提取及宏基因组Fosmid文库构建  

DNA Extraction and Construction of a Metagenomic Fosmid Library of Alpine Meadow Soil from the Mila Mountains in Tibet,China

作　　者：芦晓飞[1] 赵志祥[2] 谢丙炎[1] 杨宇红[1] 冯东昕[1] 

机构地区：[1]中国农业科学院蔬菜花卉研究所,北京100081 [2]湖南农业大学生物安全科技学院,长沙410128

出　　处：《应用与环境生物学报》2009年第6期824-829,共6页Chinese Journal of Applied and Environmental Biology

基　　金：国家重点基础研究发展计划项目(No.2009CB119000);公益性行业(农业)科研专项经费项目(No.nyhyzx07-050)资助~~

摘　　要：青藏高原土壤中富含特殊的微生物资源,因大多数未能得到培养而无法利用.提取微生物总DNA及构建宏基因组文库的方法是研究未培养微生物的重要方法(免培养法).西藏米拉山高寒草甸土壤中腐植酸、有机质含量非常高,DNA提取非常困难,本研究表明直接法提取该样品的DNA片段小,杂质含量高,不能满足构建大插入片段文库的要求;结合低速离心和Nycodenz密度梯度离心先分离出微生物细胞的间接法虽然DNA产率降低,但是纯度高,片段长,多样性丰富,更适合于构建大插入片段文库.在间接法提取高质量西藏高寒草甸土壤微生物DNA的基础上,成功构建了一个包含30624个克隆、库容量超过1Gbz稳定性好的宏基因组Fosmid文库,为从西藏高原土壤中挖掘和利用新的功能基因研究奠定了基础.The Qinghai-Tibetan Plateau represents an extreme environment for special microbial resources. Extraction of total microbial DNA with cultured-in-dependent method and construction of metagenomic library is an alternative method to exploit uncultured microorganisms. The meadow soil from the Mila Mountains in Tibet was selected as alpine soil type and was collected. A pre-experiment indicated that the soil had high organic matter and humic acid content,which resulted in a great difficulty in DNA extraction. Our results showed that the DNA obtained by direct extraction from the soil was in small DNA fragments with high contamination, and it was not suitable for constructing big insert fragment library. Comparison with direct extraction, the DNA extracted from cells separated from combing low speed centrifugation and Nycodenz gradient centrifugation had larger molecular weigh and higher purity with great diversity. A microbial metagenomic library, which contained 30 624 clones and a total genomic storage capacity over 1 Gb, was constructed with the DNA fragments from indirect method. Bioassay demonstrated that the constructed Fosmid library had large capability of insert DNA with unknown species. The method for constructing library provides a powerful tool for soil samples from extreme environment and the constructed library provides resources for finding and utilizing new functional genes from the Tibetan Plateau. Fig 7, Tab 3, Ref27

关 键 词：西藏 米拉山 高寒草甸 土壤微生物 宏基因组 FOSMID文库 

分 类 号：S812.2[农业科学—草业科学]