3 次连续重复提取DNA 能较好反映土壤微生物丰度

【目的】研究同一个土壤需要反复提取几次才能在最大程度上反映土壤微生物的丰度,探讨风干土壤代替新鲜土壤用于微生物丰度研究的可行性。【方法】针对两种理化性质具有较大差异的旱地和稻田新鲜土壤及其风干土壤,分别对土壤微生物进行5次连续裂解提取DNA。通过实时荧光定量PCR技术分析连续反复提取对土壤古菌和细菌16S rRNA gene数量、氨氧化古菌和细菌功能基因amoA数量的影响。【结果】3次连续提取DNA占5次提取DNA总量的76%以上,氨氧化古菌、氨氧化细菌、古菌和细菌4类微生物的3次连续提取最低回收率为77.5%;与新鲜土壤相比,风干处理导致氨氧化古菌、氨氧化细菌、古菌、细菌的数量分别降低84.3%、81.2%、12.5%和90.3%,然而,2种土壤风干过程中主要微生物类群的数量变化规律基本一致,表明土壤微生物对风干处理的响应可能受土壤类型的影响较小。【结论】土壤微生物连续3次裂解能较好反映微生物丰度。与新鲜土壤相比,风干过程显著降低了土壤微生物丰度,然而,通过风干土壤中微生物丰度的变化趋势反映新鲜土壤中微生物数量变化规律具有一定的可行性。

Three successive extractions reduce quantification bias of microbial communities associated with incomplete DNA recovery in soil

[Objective] To assess the quantification bias associated with incomplete extractions of soil microbial DNA and the feasibility of air-dried soil for microbial ecology study.[Methods] The flooded rice soil and upland wheat soil were used,and multiple extractions of soil microbial DNA was performed by lysing a single sample for 5 successive times.The copy number of 16S rRNA and amoA genes of Archaea and Bacteria was quantified in each DNA extraction by real-time PCR.[Results] Cumulative DNA yields in 3 successive extractions accounted for more than 76% of microbial DNA in soils,and more than 77.5% of gene copies could be recovered.Air-drying decreased the abundance of bacterial 16S rRNA gene and archaeal 16S rRNA gene by 90.3% and 12.5%,and the abundance of bacterial and archaeal amoA genes showed a decline by 81.2% and 84.3%,respectively.The decline showed similar trend in two soils,suggesting air-dried soil could be of choice for biogeographic survey of microbial communities.[Conclusion] Three successive extractions of microbial DNA in soil could be of choice for microbial ecology study in order to reduce quantification bias associated with incomplete DNA recovery.Air-dried soil could be employed under certain circumstances,and further investigation is warranted for the underlying mechanism by which microbial communities manage to survive the desiccation of soil.