用于分子生态学研究的土壤微生物DNA提取方法

应用生态学报 ›› 2005, Vol. 16 ›› Issue (5): 956-960.

用于分子生态学研究的土壤微生物DNA提取方法

张于光^1,2, 李迪强¹, 王慧敏², 肖启明²

1. 中国林业科学研究院森林生态环境与保护研究所, 北京 100091;
2. 湖南农业大学生物安全科技学院, 长沙 410128

收稿日期:2004-02-24 修回日期:2004-07-02 出版日期:2005-05-15 发布日期:2005-05-15
通讯作者: 李迪强
基金资助:
国家"十五"科技攻关项目(2001BAS10B10)和国家林业局重点资助项目.

Extraction method of soil microbial DNA for molecular ecology study

^1,2

1. Institute of Forestry Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China;
2. College of Biosafety Science and Technology, Hunan Agricultural University, Changsha 410128, China

Received:2004-02-24 Revised:2004-07-02 Online:2005-05-15 Published:2005-05-15

摘要/Abstract

摘要： 利用SDS高盐法和变性剂加SDS高盐法对土壤微生物总DNA进行了提取,然后通过电泳加树脂柱回收和连续2次树脂柱回收方法进行了纯化.结果表明,变性剂加SDS高盐法的DNA提取效率明显高于前者,电泳加树脂柱法的纯化效果更好.通过PCR扩增表明,经过纯化后的DNA,都可以进行16SrDNA扩增和nirK、nosZ、nifH等功能基因的扩增.因此,变性剂加SDS高盐法是一种更为高效、可靠且适合于环境微生物分子生态学研究的DNA提取方法.

关键词: 分子生态学, 土壤微生物, DNA提取

Abstract: In environmental microbiology,molecular ecology study has been widely concerned in the world,while high quality DNA is the basis of the study.In our study,soil microbial DNA was extracted by the methods of SDS lysis and denaturant plus SDS lysis,and purified with gel electrophoresis plus minicolumn and double minicolumn methods.The results showed that denaturant plus SDS lysis could extract DNA more efficiently,and gel electrophoresis plus minicolumn could help to obtain purer DNA that was available in amplifying its 16S rDNA and functional genes by PCR.Therefore,denaturant plus SDS lysis could be an efficient and reliable method to extract DNA in molecular ecology studies.

Key words: Molecular ecology, Soil microorganism, DNA extraction

中图分类号:

S154.4

张于光, 李迪强, 王慧敏, 肖启明. 用于分子生态学研究的土壤微生物DNA提取方法[J]. 应用生态学报, 2005, 16(5): 956-960.

参考文献

[1] Abed MM, Safi MD, Koster J, et al. 2002. Microbial diversity of a heavily polluted microbial mat and its community changes following degradation of petroleum compounds. Appl Environ Microbiol, 68(4):1674～1683
[2] Chen H(陈灏),Tang X-S(唐小树),Lin J(林洁),et al.2002. Community constitute and phylogenetic analysis on soil uncultured microorganism. Acta Microbiol Sin (微生物学报), 42(4):478 ～483 (in Chinese)
[3] Christopher R, Alexander M, Hermann B. 2002. Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil. Appl Environ Microbiol, 68 (8):3818～3829
[4] Colin RJ, Jennifer PH, Dache W, et al. 1997. A simple, efficient method for the separation of humic substances and DNA from environmental samples. Appl Environ Microbiol, 63 (12):4993 4995
[5] Gao P-P(高平平), Zhao L-P(赵平). 2002. DNA extraction from activated sludge for molecular community analysis. Acta EcolSin(生态学报),22(11) :2015～2019(in Chinese)
[6] Gesche B, Andreas F, Karl-Paul W. 1998. Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples.Appl Environ Microbiol, 64(10):3769 ～3775
[7] Holben WE, Jansson JK, Chelm BK, et al. 1988. DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl Environ Microbiol, 54:703～711
[8] Liesack W, Stackebrandt E. 1992. Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J Bacteriol, 174:5072～5078
[9] Lomans BP, Luderer R, Peter S, et al. 2001. Microbial populations involved in cycling of dimethyl sulfide and methanethiol in freshwater sediments. Appl Environ Microbiol, 67(3):1044 ～1051
[10] Lovell CR, Piceno Y. 1994. Purification of DNA from etuarine sediments. J Microbiol Methods, 20:161 ～174
[11] Malik M, Kain J, Pettigrew C, et al. 1994. Purification and molecular analysis of microbial DNA from compost. J Microbiol Methods,20:183 ～196
[12] Mehta MP, Butterfield DA, Baross JA. 2003. Phylogenetic diversity of nitrogenase(nifH)genes in deep-sea and hydrothermal vent environments of the Juan de Fuca Ridge. Appl Environ Microbiol, 69(2) :960～970
[13] Ogram A, Sayler GS, Barkay T. 1987. The extraction and purification of microbial DNA from sediments. J Microbiol, 7:57～66
[14] Philippot L, Piutti S, Martin LF, et al. 2002. Molecular analysis of the nitrate-reducing community from unplanted and maize-planted soils. Appl Environ Microbiol, 68(12) :6121 ～6128
[15] Picard C, Ponsonnet C, Paget E, et al. 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl Environ Microbiol, 58:2717～2722
[16] Porteous LA, Armstrong JL. 1991. Recovery of bulk DNA from soil by a rapid, small-scale extraction method. Curr Microbiol, 22:345～348
[17] Richard AH, Qiu XY, Wu LY, et al. 2001. Simultaneous recovery of RNA and DNA from soils and sediments. Appl Environ Microbiol, 67(10):4495 ～4503
[18] Smalla K, Cresswell N, Mendonca-Hagler LC, et al. 1993. Rapid DNA extraction protocol from soil for polymerase chain reactionmediated amplification. Appl Environ Microbiol, 74:78 ～85
[19] Steffan RJ, Atias RM. 1988. DNA amplification to enhance detection of genetically engineered bacteria in environmental samples.Appl Environ Microbiol, 54:2185～2191
[20] Steffan RJ, Goksoyr J, Bej AK, et al. 1988. Recovery of DNA from soils and sediments. Appl Environ Microbiol, 54:2908 ～2915
[21] Tian C-J(田春杰),Chen J-K(陈家宽),Zhong Y(钟扬).2003.Phylogenetic diversity of microbes and its perspectives in conservation biology. Chin J Appl Ecol (应用生态学报), 14(4) :609 612(in Chinese)
[22] Torsvik V, Goksoyr J, Daae FL. 1990. High diversity in DNA of soil bacteria. Appl Environ Microbiol, 56:782～787
[23] Tsai YL, Olson BH. 1991. Rapid method for direct extraction of DNA from soil and sediments. Appl Environ Microbiol, 57:1070～1074
[24] Vesico PA, Nierzwicki SA. 1995. Extraction and purification of PCR amplifiable DNA from lacustrine subsurface sediments. J Microbiol Methods, 21:225～233
[25] Ward DM, Weller R, Bateson MM. 1990.16S rRNA sequences reveal numerous uncultured inhabitants in a natural community. Nature, 345:63 ～65
[26] William GW, Susan MB, Dale AP, et al. 1991.16S Ribosomal DNA amplification for phylogenetic study. J Bacteriol, 173 (2):697 ～703
[27] Xiang W-S(向万胜), Wu J-S(吴金水), Xiao H-A(肖和艾), et al. 2003. Advances in extraction and purification of soil microorganism. Chin J Appl Ecol(应用生态学报), 14(3) :453～456(in Chinese)
[28] Yeates C, Gillings MR, Davison AD, et al. 1997. PCR amplification of crude microbial DNA extracted from soil. Lett Appl Microbiol,25:303 ～307
[29] Yeates C, Gillings MR, Davision AD, et al. 1998. Methods for microbial DNA extraction from soil for PCR amplification. Biol Proced Online, 1(1) :40～47
[30] Zhang H-W(张慧文),Zhang Q-R(张倩如),Zhou Q-X(周启星),et al. 2003. Introduction and progress of molecular microbial ecology. Chin J Appl Ecol (应用生态学报), 14(2) :286～292(in Chinese)
[31] Zhong M(钟鸣),Zhou Q-X(周启星).2002. Molecular ecological technology of microorganisms and its application to research on environmental pollution. Chin J Appl Ecol (应用生态学报), 13(2):247 ～251 (in Chinese)
[32] Zhou JZ, Mary AB, Tiedje JM. 1996. DNA recovery from soils of diversity composition. Appl Environ Microbiol, 62(2) :316～322