浙贝母土壤真菌DNA提取方法的优化

摘要

[目的]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往往比较困难,本实验通过方法优化以寻找适合浙贝母土壤真菌DNA的最佳方法.[方法]分别采用传统玻璃珠法,Ultra CleanTM土壤DNA提取试剂盒法及改进的试剂盒法等3种方法,提取宁波浙贝母种植区的根际土壤微生物总DNA,利用巢式PCR扩增真菌DNA片段.[结果]3种方法均可提取土壤微生物总DNA.其中改进的试剂盒法提取的DNA电泳结果条带最清晰,试剂盒法提取的DNA电泳结果次之,传统玻璃珠法提取的DNA电泳条带不清晰,且有拖尾.对上述三种方法所提取的DNA做巢式PCR分析真菌特异性,传统方法电泳无目的条带,试剂盒法只有第一次PCR电泳有目的条带,只有改进的UltracleanTM土壤DNA提取试剂盒法最终获得了350 bp左右的目的条带,而且利用变性凝胶梯度电泳技术(Denatured gradient gel electrophoresis,DGGE)成功地检测到不同种源以及根际与非根际土中微生物的差异.因此认为改进的试剂盒法为提取土壤真菌DNA的最优选择.[结论]在土壤真菌DNA提取中,合适的振荡方法是土壤真菌能否破壁的关键性因素.%[Objective]It is difficult to isolate fungi DNA from the soil just because of the complex properties of soil and the special nature of fungal cell wall. [Method] Three DNA extraction methods were done to explore the best method for fungi, traditional glass bead method, Ultraclean? soil DNA isolation Kit and improved UltracleanTM soil DNA isolation Kit. [Result) Soil DNA fragments(about 9400-23000bp) could be obtained using these three methods. The band was the clearest using improved DNA kit, followed by the DNA kit, and the band was the lightest with a tail section using traditional method. Only using modified kit could PCR band(about 350bp) be obtained and it showed that the differences of fungal diversity and stucture among different provenances could be domentrated clearly by DGGE. [Conclusion] Among these three methods, the improved UltracleanTM soil DNA extraction kit method was the optimal choice for fungi analysis, indicating that oscillation method was a critical factor for fungal DNA isolation.