紫色水稻土水稳性团聚体土壤微生物基因组DNA提取方法探讨

为了找到提取土壤微生物总DNA的最佳方法,通过OD值检验、凝胶电泳、PCR和DGGE分析,比较了Reddy法、基于DNAout kit试剂盒改进的实验方法、以及Kuske修订法、Edgcomb改进法、SDS高盐提取法、Eichner调整法等常用的不同土壤微生物基因组的DNA提取方法在亚热带地区长期免耕紫色水稻土水稳性团聚体0.25～2.0 mm粒径上的提取效果.结果表明,6种方法都可以从团聚体中提取到长度大于23.1 kb的DNA片段,但不同方法提取的DNA的产量存在明显差异,土壤总DNA均不需纯化就可以用于PCR扩增,使用细菌16S rDNA基因V3区的通用引物可扩增得到相应的片段.研究表明,改进的DNAout kit试剂盒法是长期免耕紫色水稻土水稳性团聚体中微生物基因组DNA的最佳提取方法.

Microbial DNA Extraction Method on Soil Water-stable Aggregates about the Purple Paddy Soil

The objective of the study is to get a suitable method of DNA extraction for various types of soil. Subtropical areas of long-term no-tillage purple rice soil water stability 0. 25～2. 0 mm diameter was choosed as experiment material and the effect of DNA extraction was detected by Gel electrophoresis, OD(optical density),PCR (Polymerase Chain Reaction) and DGGE (denaturing gradient gel electrophoresis). Six protocols choosed in the study included Reddy method,modified DNAout kit protocol, improved Kuske revised method, improved Edgcomb method, SDS high salt extraction method, and Eichner of DNA extraction method. The results were as followed: six protocols could get all DNA fragment of about 23.1 kb in the soil aggregate size, and DNA extracted by all protocols could be used as templates for PCR amplification using primers of the bacterial 16SrDNA V3 gene without purification. However, the yields among different methods were different. These results inferred that modified DNAout kit protocol is the most suitable for PCR amplification and DGGE extracted microbial DNA from the no-tillage purple paddy soil.