摘要
环境微生物宏基因组是一个巨大的基因资源库,但是仅有0.1%-1%的微生物在现有技术条件下是可培养的,因此致使未培养微生物基因资源的开发利用受到限制。宏基因组技术直接提取环境样品总DNA,避开了微生物分离培养的问题,极大扩展了微生物资源的利用空间,增加了获得新生物活性物质的机会。简要介绍了宏基因组的概念及宏基因组克隆技术的基本操作流程和技术要点,重点阐述了目的基因富集、核酸提取、载体和宿主系统选择、宏基因组文库筛选等“瓶颈”技术的研究进展。目的基因富集技术主要包括稳定同位素探针（SIP）、抑制消减杂交（SSH）和差异显示（DD）等。基因文库筛选分为序列依赖性筛选和非序列依赖性筛选,其中序列依赖性筛选包括特定基因PCR、反转录PCR（RT-PCR）、DNA微阵、亲和捕获等技术;非序列依赖性筛选主要指基于基因表达活性筛选和基因“陷阱”技术等。此外,介绍了一些近年来通过构建宏基因组文库筛选目的基因的应用实例。
Microbial metagenome is the largest gene reservoir in environment. Exploring uncultured microbial gene resource has been restricted, due to the fact that only 0.1%-- 1% of the microorganisms can be cultivated using current techniques. Metagenome libraries can be constructed by direct extracting DNA from environmental samples to avoid the limitations of cuhure-dependent methods. Metagenomic technique has greatly enhanced the utilization of microbial resource with more opportunity of obtaining novel bioactive compounds. In this paper, the concept of metagenome, basic operation process and some key technical details are introduced. The introduction is focused on the advancement in some ＂ bottle neck＂ techniques, including enrichment of interested genes, extraction of nucleic acid, selection of vector and host system, and metagenomic library screening. Gene enrichment involves techniques such as Stable Isotope Probing （SIP） , Suppression Subtractive Hybridization （SSH）, and Differential Display （DD）. Metagenomic library screening includes sequence- dependent and sequence-independent method. The former method includes gene-specific Polymerase Chain Reaction （ PCR）, reverse transcription PCR （RT-PCR） , DNA microarray, affinity capture, and so on. The latter method is mainly referred as activity-based screening and ＂ gene trap ＂ techniques. Some recent examples of gene targeting through metagenome libraries are also introduced.
出处
《生态学报》
CAS
CSCD
北大核心
2008年第4期1762-1773,共12页
Acta Ecologica Sinica
基金
国家重点基础研究发展计划资助项目(2004CB418505)
国家自然科学基金资助项目(30670391)~~
关键词
宏基因组
未培养微生物
核酸提取
宏基因组文库构建
宏基因组文库筛选
Metagenome
Uncultured microorganisms
Nucleic acid extraction
Metagenomic library construction
Metagenomic library screening
