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[1]芦晓飞,赵志祥,谢丙炎,等.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[J].应用与环境生物学报,2009,15(06):824-829.[doi:10.3724/SP.J.1145.2009.00824]
　LU Xiaofei,ZHAO Zhixiang,XIE Bingyan,et al.DNA Extraction and Construction of a Metagenomic Fosmid Library of Alpine Meadow Soil from the Mila Mountains in Tibet, China[J].Chinese Journal of Applied & Environmental Biology,2009,15(06):824-829.[doi:10.3724/SP.J.1145.2009.00824]
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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()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
15卷
期数:
2009年06期
页码:
824-829
栏目:
研究论文
出版日期:
2009-12-25

文章信息/Info

Title:
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; 赵志祥; 谢丙炎; 杨宇红; 冯东昕
（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）
Author(s):
LU Xiaofei;  ZHAO Zhixiang;  XIE Bingyan;  YANG Yuhong;  FENG Dongxin
(1Institute of Vegetables and Flowers, Chinese Academy of Agricultural Science, Beijing 100081, China)
(2College of Bio-safety Science and Technology, Hunan Agriculture University, Changsha 410128, China)
关键词:
西藏; 米拉山; 高寒草甸; 土壤微生物; 宏基因组; Fosmid文库
Keywords:
Tibet;  Mila Mountains;  alpine meadow;  soil microorganism;  metagenomic;  Fosmid library
分类号:
S154.3 (27)
DOI:
10.3724/SP.J.1145.2009.00824
文献标志码:
A
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30 624个克隆、库容量超过1 Gb、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. 图7 表3 参27
Abstract:
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, Ref 27

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备注/Memo

备注/Memo:
国家重点基础研究发展计划项目（No. 2009CB119000）和公益性行业（农业）科研专项经费项目（No. nyhyzx07-050）资助 Supported by the State Key Basic Research and Development Program of China (No. 2009CB119000), and the Public Industry (Agriculture) Specific Research Program of China (No. nyhyzx07-050)
更新日期/Last Update: 2009-12-24