Abstract
An effective method for plant rhizosphere microbial genomic DNA extraction was established. High-purity nucleic acids extracted from soil samples, with considerable yield, could be used for study of soil microorganism molecular ecology. Genomic DNAs were extracted from 12 soil samples of different crop roots. Above 10 μg of genomic DNA with approximately 20-kb fragment length was isolated from 1 g of soil sample. The average value of A260/A230 of the genomic DNA was 1.505, while that of A260/A280 was 1.780. The absorption curves of the full wavelengths of the DNA extracted were consistent with that of pure nucleic acid. DNAs from different dilutions of pollutants were applicable for restriction enzyme digestion analysis, and the lambda DNA in 100-fold diluted soil samples had the same restriction enzyme digestion results as done in ddH2O. Amplicons was produced with the expected molecular size by polymerase chain reaction (PCR).Therefore, the DNA extracted by this method was suitable to be used in analysis of bacterial ecology composition by denaturing gradient gel electrophoresis. Key words: DNA extraction, DNA quality, PCR amplification, soil microbial community diversity, DGGE.
Highlights
Soil is a complex environment, and is a major reservoir of microbial genetic diversity (Robe et al, 2003)
We developed a standard procedure for the effective quantification and purification of metagenomic DNA from the soil around plant roots, amenable to PCRDGGE based analysis to evaluate microbial community diversity
A260/A230 of the pure nucleic acid was 1.820, pure humus was 0.8; and the average A260/A230 of the genomic DNA extracted from different soil samples was 1.505, which is close to the ratio of pure nucleic acids
Summary
Soil is a complex environment, and is a major reservoir of microbial genetic diversity (Robe et al, 2003) Soils and their microbial inhabitants are critical to global biogeochemical cycles including carbon, nitrogen and phosphorus, which support all other forms of terrestrial diversity. Because of their importance on multiple levels, soils have been the subject of studies in microbial ecology for decades (Borneman and Triplett, 1997; Nunanet al., 2003; Skinner et al, 1952; Skyring and Quadling, 1969; Steffanet al., 1988; Waksman and Woodruff, 1940; Williamson et al, 2010). The type and amount of nutrients released by plants will affect both the numbers of organisms and their diversity, for example, insecticidal Cry proteins derived from insect-resistant Bt crops in soils through cultivation of Bt crops (Atwood, 2011; Birch et al, 2007; Dauduet al., 2009; Dunfield and Germida, 2001; Griffiths et al, 2007; Groot and Dicke, 2002; Heckmannet al., 2006; Höss et al, 2011)
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