堆肥中微生物总DNA的高效提取  

An efficient method for DNA extraction from compost

作　　者：何丽鸿[1] 赵勇[1] 陈明杰[2] 潘迎捷[1] 

机构地区：[1]南京农业大学生命科学院微生物学系,南京210095 [2]上海市农业遗传育种重点开放实验室上海市农业科学院食用菌研究所,上海201106

出　　处：《微生物学报》2006年第1期162-165,共4页Acta Microbiologica Sinica

摘　　要：采用化学裂解和酶解相结合的方法,选择加入PVPP的高盐缓冲液作为细胞裂解的反应体系,并以PEG-8000进行DNA沉淀,从高有机含量的堆肥样品中进行微生物总DNA的提取。结果表明,从4种性质不同的堆肥中均获得了高质量的微生物总DNA,所得的DNA分子片段在23kb左右;每克干重堆肥的总DNA提取量为63.54±12.08μg^106.50±28.36μg,A260/A280大于1.6,A260/A230大于1.8,不用经过纯化可以直接进行PCR扩增和限制性酶切;以该DNA为模板进行微生物区系的DGGE分析,显示了丰富的微生物多样性。该方法减少了通常环境样品DNA提取过程中的纯化步骤,减少了DNA的损失,为从事微生物分子生态学,尤其是那些针对高有机含量以及获取极为不易的环境样品的研究而言是十分有益的。An efficient method for obtaining DNA from compost which contained high levels of organic matter was developed. The protocol consisted of washing with phosphate-EDTA before extraction, cell lysis with hot-SDS and enzymes （lysozyme, lywalzyme, proteinase K）, removing humic acid and other inhibitors with PVPP and precipitation with PEG-8000. The compost total DNA was extracted from four different composts, the DNA yield was 63.54 ± 12.08 - 106.50±28.36μg/g of dry compost. Molecular size of DNA obtained using this protocol was about 23kb and contained low protein and humic acid contamination with the A260/A280 ratios exceeding 1.6 and A260/A230 ratios reaching 1.8. Usually, additional purification steps such as agarose gel electrophoresis, gel permeation chromatography, or affinity chromatography were needed to get PCR-amplifiable DNA , but the DNA obtained using this protocol could directly be used to PCR-amplification and restriction enzyme digestion. Just like purity of DNA template, lower DNA yield also appears to introduce a bias towards lower community diversity. In this study compared the purified DNA the direct DNA reveals higher microbial community diversity assessed by denaturing gradient gel electrophoresis（DGGE） of amplified V3 region of 16S rDNA.

关 键 词：堆肥 DNA的高效提取 高有机含量 DGGE 

分 类 号：Q781[生物学—分子生物学] S141.4[农业科学—肥料学]