摘要
真菌的细胞壁,增加了提取基因组DNA的难度,为找到满足竹黄菌全基因组序列分析的高质量DNA的提取方法,分别使用改良CTAB法、蛋白酶K法和高盐沉淀法提取竹黄菌菌丝体的基因组DNA,并进行琼脂糖凝胶电泳、紫外分光光度计测定浓度和纯度、ITS序列的PCR扩增和酶切检测。结果表明:3种方法提取等量竹黄菌菌丝体的基因组DNA,蛋白酶K法和高盐沉淀法均能得到较为完整的且大于15kb的DNA,改良CTAB法DNA发生降解;通过PCR和酶切检测,3种方法所得基因组DNA均能有效地进行相应的酶反应。综合考虑基因组DNA的浓度、纯度、完整性和有无降解等,蛋白酶K法提取得到的基因组DNA(浓度为233.495ng/μL,OD_(260)/OD_(280)为1.806)最适用于全基因组的序列分析。
Unique fungal cell walls increase the difficult of extracting genomic DNA.In order to find a suitable DNA extracting method for the whole genomic sequence analysis of S.bambusicola,the modified CTAB method,Proteinase K method and the high-concentration-salt precipitation were compared.The DNA concentration and purity were determined by using agarose gel electrophoresis and ultraviolet spectrophotometer.PCR amplification of the ITS sequence as well as enzyme detection were also compared.Results：Proteinase K method and the high-concentration-salt precipitation obtained more complete and 〉15kb DNA.However,DNA extracted by the modified CTAB degrades.Meanwhile,by PCR and enzyme detection,the genomic DNA obtained by three methods can effectively participate in corresponding enzyme reaction.Eventually,based on the consideration of concentration,purity,integrity and degradation of DNA,the genomic DNA（concentration is 233.495 ng/μL,OD_（260）/OD_（280） is 1.806）extracted by Proteinase K method was the most applicable for the genome sequence analysis.
出处
《贵州农业科学》
CAS
2015年第12期19-22,共4页
Guizhou Agricultural Sciences
基金
国家自然科学基金项目"竹黄菌侵染桂竹互作机制研究"(NSFC 31460199)
国家自然科学基金项目"西南地区竹黄遗传多样性与寄主专化性研究"(31160160)
关键词
竹黄菌
改良CTAB法
蛋白酶K法
高盐沉淀法
全基因组测序
Shiraia bambusicola
modified CTAB method
Proteinase K method
high-concentration salt precipitation
whole genome sequencing
