4种不同土壤微生物DNA提取方法对DGGE分析微生物群落的影响

福建农业学报 ›› 2012, Vol. 27 ›› Issue (4): 367-372.

4种不同土壤微生物DNA提取方法对DGGE分析微生物群落的影响

吕新^1,2, 陈丽华^1,2, 李玥仁^1,2

1. 福建省农业科学院中心实验室,福建福州 350003;
2. 精密仪器农业测试重点公共实验室,福建福州 350003

收稿日期:2012-03-23 修回日期:2012-04-15 出版日期:2012-04-30 发布日期:2012-04-30
通讯作者: 李玥仁(1966-),男,研究员,博士,主要从事生物多样性研究(E-mail:yuerenli@yahoo.com.cn)
基金资助:
农业部转基因生物新品种培育科技重大专项(2009ZX08011-029B)

Effect of DNA Extraction on DGGE Analysis of Microbial Community in Soil

LV Xin^1,2, CHEN Li-hua^1,2, LI Yue-ren^1,2

1. Central Laboratory, Fujian Academy of Agricultural Science, Fuzhou,Fujian 350003, China;
2. Key Public Precision Testing Laboratory for Agricultural Applications, Fuzhou,Fujian 350003, China

Received:2012-03-23 Revised:2012-04-15 Online:2012-04-30 Published:2012-04-30

摘要/Abstract

摘要： 分别应用高盐法、玻璃珠破碎法、冻融法和试剂盒法4种不同土壤微生物DNA提取方法提取水稻稻田土壤微生物DNA,并通过细菌16S rRNA V₃区通用引物GC338f-530R和真菌18S rRNA特异性引物NS1-GCFung进行PCR扩增结合变性梯度凝胶电泳(CGGE)分析,对4种DNA提取方法进行评价。结果表明,玻璃珠破碎法和试剂盒法提取的DNA均能满足土壤微生物多样性分析的要求;其中试剂盒方法操作简单,提取的DNA 质量较高,但DNA 产量较低且成本昂贵;玻璃珠破碎法用时较长,步骤繁琐,DNA产量较高,纯度较低,但对后续PCR扩增和DGGE分析没有明显影响,且成本低廉。

关键词: 土壤微生物, DNA提取, 变性梯度凝胶电泳

Abstract: The Zhou, Martin, Bourrain and Kit methods were used to extract microbial DNA in paddy soil. They were evaluated based on the amplification of bacterial 16S rRNA V₃ variable region amplified by the primer pair GC338f-530R and the fungal 18S rRNA region amplified by the primer pair NS1-GCFung and the subsequent DGGE analysis of amplified products. The results showed that the Martin and the Kit methods satisfied the analytical requirements of soil microbial diversity. The Kit method was simpler and faster, and the quality of its extracted DNA higher, but the yield and purity of DNA extracted were lower and the cost greater than the Martin method. However, the deficiencies did not appear to impose significant negative effect on the PCR amplification and DGGE analysis that followed.

Key words: soil microorganisms, DNA extraction, DGGE

中图分类号:

S 154.36

吕新, 陈丽华, 李玥仁. 4种不同土壤微生物DNA提取方法对DGGE分析微生物群落的影响[J]. 福建农业学报, 2012, 27(4): 367-372.

LV Xin, CHEN Li-hua, LI Yue-ren. Effect of DNA Extraction on DGGE Analysis of Microbial Community in Soil[J]. Fujian Journal of Agricultural Sciences, 2012, 27(4): 367-372.

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