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用于分子生态学研究的堆肥DNA提取方法
引用本文:杨朝晖,肖勇,曾光明,刘云国,邓久华.用于分子生态学研究的堆肥DNA提取方法[J].环境科学,2006,27(8):1613-1617.
作者姓名:杨朝晖  肖勇  曾光明  刘云国  邓久华
作者单位:湖南大学环境科学与工程学院,长沙,410082
基金项目:国家高技术研究发展计划(863)项目(2004AA649370);国家自然科学基金项目(50478053);湖南省自然科学基金项目(04JJ3004)
摘    要:分子生态学为堆肥微生物的研究提供了新的技术手段,DNA的提取是该技术的基础,但由于腐殖酸类物质的污染,增加了堆肥微生物总DNA的提取难度.采用了3种不同的方法(溶菌酶法、超声波破碎法和蛋白酶K-CTAB法)从堆肥中提取微生物的总DNA,使用核酸和蛋白质分析仪检测后表明3种提取方法获得的DNA产量均较高;琼脂糖凝胶电泳结果表明其长度约为23 kb;使用细菌16S rRNA基因通用引物(27F和1 495R)对总DNA进行PCR扩增,都获得了几乎全长的16S rDNA序列(约1.5 kb);利用限制性内切酶(Hae Ⅲ和AluⅠ)对纯化后的PCR产物进行RFLP分析,结果表明3种方法提取的DNA反映了比较一致的微生物多样性.虽然3种方法各有优缺点,但其提取的DNA都可以用于堆肥微生物的分子生态学研究,可以根据实际需要选用某一种方法用于提取堆肥总DNA.

关 键 词:堆肥  分子生态学  16SrRNA  DNA提取  RFLP
文章编号:0250-3301(2006)08-1613-05
收稿时间:9/1/2005 12:00:00 AM
修稿时间:2005-09-012005-11-03

DNA Extraction Methods of Compost for Molecular Ecology Analysis
YANG Zhao-hui,XIAO Yong,ZENG Guang-ming,LIU Yun-guo and DENG Jiu-hua.DNA Extraction Methods of Compost for Molecular Ecology Analysis[J].Chinese Journal of Environmental Science,2006,27(8):1613-1617.
Authors:YANG Zhao-hui  XIAO Yong  ZENG Guang-ming  LIU Yun-guo and DENG Jiu-hua
Institution:College of Environmental Science and Engineering, Hunan University, Changsha 410082, China. yzh@hnu.cn
Abstract:Molecular ecology provides new techniques for studying compost microbes, and the DNA extraction is the basis of molecular techniques. Because of the contamination of humic acids, it turns to be more difficult for compost microbial DNA extraction. Three different approaches, named as lysozyme lysis, ultrasonic lysis and proteinase K lysis with CTAB, were used to extract the total DNA from compost. The detection performed on a nucleic acids and protein analyzer showed that all the three approaches produced high DNA yields. The agarose gel electrophoresis showed that the DNA fragments extracted from compost had a length of about 23 kb. A eubacterial 16S rRNA gene targeted primer pair (27F and 1 495R) was used for PCR amplification, and all the samples got almost the full length 16S rDNA sequence (about 1.5 kb). After digested by restriction endonucleases (Hae Ill and Alu I), the restriction map showed relatively identical microbial diversity in the DNA, which was extracted by the three different approaches. All the compost microbial DNA extracted by the three different approaches could be used for molecular ecological study, and researchers should choose the right approach for extracting microbial DNA from compost based on the facts.
Keywords:compost  molecular ecology  16S rRNA  DNA extraction  RFLP
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