摘要
由于土壤理化性质的复杂性和真菌细胞壁结构的特殊性,从土壤样品中提取真菌基因组DNA比较困难。中国北方土壤与其它地区土壤相比有其自身的特点,因此,有必要优化一种适合于北方土壤真菌DNA提取的方法。本实验向灭菌黑土中分别投加12种在系统分类上差别较大的真菌,以传统土壤总DNA提取方法及纯菌DNA提取方法为基础,分别与蜗牛酶,纤维素酶进行组合、优化,得到7种不同的土壤真菌基因组DNA提取方法。利用真菌28SrDNA通用引物U1/U2-GCPCR-DGGE分析方法分别考察了7种不同方法所提取土壤真菌基因组DNA的多样性和代表性。结果表明:1)液氮研磨,纤维素酶、蜗牛酶和溶菌酶(浓度分别为6、3和1mg.ml-1)37℃作用60min,2%SDS于65℃裂解30min;2)-65℃～65℃冻融3次,纤维素酶、蜗牛酶和溶菌酶(浓度分别为6、3和1mg.ml-1)37℃作用180min,2%SDS于65℃裂解30min的组合具有较好的提取效果。利用后一种方法分别对3种理化性质差异较大的中国北方自然土壤样品真菌DNA进行提取并分析,表明所提取土壤基因组DNA真菌特异性PCR-DGGE图谱条带丰富,该方法可用于多种北方土壤真菌多样性研究。
Owing to the complexity of soil physical and chemical properties and the structural particularity of fungal cell wall, the extraction of fungal DNA is more difficult than that of bacterial DNA from soil samples. The soils in North China have their own special properties, compared with the others. Therefore, it is necessary to optimize the extraction method of fungal DNA from soils in North China to study their fungal diversity and composition. Based on the 28S rDNA- PCR-DGGE analysis with fungal universal primers, seven extraction methods were evaluated by adding twelve fungi with greater difference in taxonomy into sterilized black soil. The results showed that two methods were more efficient for the study of soil fungal diversity, i. e. , （ 1 ） grinding soil samples in liquid nitrogen, treating them with cellulase, snailase and lysozyme （6, 3, and ling · ml^-1 , respectively） for 60 minutes at 37 ℃ , and lysing with 2% SDS for 30 minutes in 65 ℃ water bath, and （2） freezing and thawing soil samples at -65 ℃ for three times, treating them with cellulase, snailase and lysozyme （6, 3 and 1 mg · ml^-1, respectively） at 37 ℃ for 180 minutes, and lysing with 2% SDS for 30 minutes at 65 ℃. The fungal DNA was extracted from three natural soils with greater differences in physical and chemical properties in North China by using the second method, and the 28S rDNA-PCR-DGGE profiles showed that the fungal DNA bands were comparatively rich, suggesting that this method could be used in fungal diversity research of North China soils.
出处
《生态学杂志》
CAS
CSCD
北大核心
2007年第4期611-616,共6页
Chinese Journal of Ecology
基金
国家重点基础研究发展规划项目(2004CB418503)
国家自然科学基金资助项目(30440031)
