用于微生物多样性分析的棉田土壤总DNA的提取

目的：探讨适用于微生物多样性研究的棉田土壤微生物总DNA提取方法。方法：采用4种方法提取不同连作和轮作处理的棉田土壤微生物总DNA,比较其纯度、产率、片段大小,并应用ARDRA技术验证其质量。结果：其中3种方法均可获得23kb的DNA片段,但不同方法提取的DNA的产率和纯度上有明显差异。改良CTAB-SDS法提取的DNA完整性好,得率为24.20μg.g-1干土,纯化后A260/A280和A260/A230为分别为1.80和1.70,纯化回收率可达70.1%,完全适用于后续的PCR分析。结论：采用该法提取棉田土壤总DNA简便而高效。对该法提取获得的棉田土壤微生物总DNA进行ARDRA和DGGE分析,所得图谱能较全面地反映不同处理间微生物多样性及群落结构的差别,为不同栽培体系下棉田土壤微生物的分子生态学研究提供了基础。

A Method of Extracting Total Microbial DNA from Cotton Soil for Microbial Diversity Analysis

Objective:The research aimed to develop a method of extracting total microbial DNA from cotton soil for microbial diversity analysis.Method: Four methods were used to extract total microbial DNA from the cotton soi of different continuou cropping and rotation treatments.The extracted DNA were evaluated comprehencively in DNA purity,yield,fragment size,and the impact on the subsequence by ARDRA and DGGE analysis of the bacterial community.Result: The DNA extracted by three methods from the eight samples were all greater than 23kb in size,but the yields and quality were different significantly.Modified CTAB-SDA method was confirmed that the yield of the extracted DNA was 24.20μg·g~(-1) dry soil.After purified,the A_(260)/A_(280) and A_(260)/A_(230) could be get 1.80,1.70 respectively ,while percent recovery of purification was 70.1%,and the extracted DNA were successfully used for the further molecular analysis.Conclusion: It was suggested that the extracted DNA could reflect the biodiversity and composition of microbial community in the soils,while the genomic fingerprinting of the DNA displayed difference.This development of the simple and highly effective DNA extraction method provided fundation for studying in molecular ecology of cotton soil microbes.