非培养方法在土壤微生物生态学研究中的应用

Culture independent methods for studies on microbial ecology of soils

由于有相当数量的土壤微生物是目前不可培养的 ,因此利用传统培养技术来研究土壤微生物 ,不仅费时费力 ,所得到的结果可能和真实的情况相差甚远。近年来发展了三类不需培养的方法来研究土壤微生物的种类和数量 ,这些方法大体上分为生物化学、生理学和分子生物学三类。生物化学方法主要根据细胞膜磷脂酸 (PLFA)的种类和数量来判定微生物的多样性 ;BIOLOG微量板分析系统是生理学方法的代表 ,它主要是根据土样细胞悬液对 95种单一碳源的利用模式来说明群落结构的变化 ;分子生物学方法是发展应用最广的方法 ,基本步骤是提取土壤的总DNA ,然后用通用引物或选择性高的引物来扩增 16SrRNA基因。由于对扩增产物分析方法的不同 ,该方法又可分为PCR DGGE ,PCR RFLP等。最近在PCR RFLP基础上发展起来的T RFLP分析方法 ,将微生物的多样性分析工作同RDP(ribosomaldatabaseproject)数据库结合 ,充分利用了Internet的数据资源共享的优势 ,具有分辨率高 ,可实现自动化等优点 ,是未来土壤微生物生态学研究的有力工具。

Since most species of microorganisms in soils are not cultivable in the laboratory, the investigation of their communities is a time-consuming and hard work by traditional culture procedure. Moreover, some wrong conclusions may be drawn. In the past decades, three kinds of culture-independent methods have been developed so that great progress has been made in this field. The biochemical method is to determinate the microbial communities by analysis of phospholipid fatty acids (PLFA) of cell membrane. The metabolism-based approach, through observations of the utilization patterns of 95 single-carbon sources performed on the BIOLOG microplates, can provide a lot of information about the microbial functional groups in the soils. The third method, molecular techniques, is most prevalently used to explore the microbial communities. Briefly, the molecular approach was started with the extraction and purification of total DNA from sample soils, followed by PCR-amplification of 16SrRNA gene with universal or special primers. As the analysis procedure of PCR products is different, a variety of approaches, for example, DGGE, RFLP and T-RFLP, have been established since 1990s. Among them, T-RFLP was developed based on the RFLP in 1997. This method combined ribosomal database project (RDP) into the analysis of microbial communities, and the number and constituents of species within a community were inferred just according to the number and intensity of the terminal restriction fragments of 16S rRNA gene. Performing T-RFLP is simple, with high resolution, and can be carried out automatically. Therefove it will play more and more important role in future research in microbial ecology of soils.