土壤微生物总DNA主要来源于胞外DNA以及活性和非活性微生物群，因此，单纯基于土壤微生物总DNA的研究无法反映出土壤中真实的活性微生物群，极易造成土壤微生物丰度及群落多样性的高估，但目前尚没有准确可行的方法筛选土壤活性微生物群。本项目拟利用PMA核酸染料具有共价不可逆结合膜受损的胞内DNA和游离DNA的技术特点，通过优化PMA预处理质量浓度、避光反应时间和曝光反应时间，建立适用于复杂土壤样品的实验操作流程；结合以RNA为核心的转录组学分析以及稳定性同位素示踪DNA-SIP技术，评价PMA用于复杂土壤样品活性微生物筛选的可行性和准确性；最后利用PMA筛选新鲜土、冷冻干燥土和自然风干土中活性微生物群，揭示活性微生物对不同土壤保存策略的适应机制。本项目的开展将为土壤活性微生物生态学研究提供新的理论和方法，同时为土壤样品保存策略及其微生物生态学中应用提供重要参考。

Soil microbial total DNA mainly originate from extracellular DNA, viable and nonviable microorganisms. Therefore, the total DNA-derived signals often lead to overestimate the viable microbial population and diversity. However, the accurate and feasible method to selectively screen viable microorganisms from complex soil matrices has not been found. Use of PMA as intercalating dyes to covalently cross-link extracellular DNA or DNA from dead cells with compromised membrane integrity, this project aims to establish standard experimental procedures for screening viable cells from complex soil matrices. PMA concentration, incubation time and exposure time will be optimized, together with transcriptomics and stable isotope probing technique applied to evaluate the feasibility and accuracy of PMA method. Further, viable microbial communities will be separated from fresh soil, freeze-dried soil and air-dried soil using PMA, and the adaptive mechanism of viable microorganisms response to different soil archived strategies will be revealed. This study will provide a new theory and method for soil viable microbial study, and important references will be developed for soil archived strategies and their application in microbial ecology.