基于GC-MS的绿僵菌代谢组学前处理技术研究  

Study on sample preparation for the metabolomics of Metarhizium based on GC-MS

作　　者：杨华[1,2] 徐金柱[2] 赵丹阳[2] 邱华龙[2] 田龙燕[2] 秦长生 YANG Hua;XU Jinzhu;ZHAO Danyang;QIU Hualong;TIAN Longyan;QIN Changsheng(College of Agriculture,South China Agricultural University,Guangzhou 510642,China;Guangdong Academy of Forestry/Guangdong Provincial Key Laboratory of Silviculture,Protection and Utilization,Guangzhou 510520,China)

机构地区：[1]华南农业大学农学院,广东广州510642 [2]广东省林业科学研究院/广东省森林培育与保护利用重点实验室,广东广州510520

出　　处：《华南农业大学学报》2021年第5期69-79,共11页Journal of South China Agricultural University

基　　金：广东省重点领域研发计划(2020B020214001)。

摘　　要：【目的】基于GC-MS技术对绿僵菌Metarhizium代谢物前处理技术进行优化,以期建立适用于绿僵菌的快速、准确的代谢组检测方法。【方法】优化绿僵菌代谢物的淬灭、提取、衍生和检测方法,测定方法的稳定性。【结果】40%(φ)冷乙醇溶液淬灭后核酸和蛋白回收率分别为9.63%和11.61%,淬灭效果优于其他淬灭剂;冷甲醇法可提取到代谢物109种,多于其他方法;衍生的时间越长,获得的代谢物越多,衍生1.5 h效果最好;气相色谱检测起始温度过高不利于代谢物的获得,50℃效果最好。最佳条件如下:40%(φ)冷乙醇溶液淬灭后用2 mL冷甲醇提取,离心后将上清液用N2吹干,加入20 mg/mL的甲氧基胺盐酸盐吡啶溶液80μL,剧烈振荡30 s后,在37℃环境中反应90 min,反应结束后冷却至室温,然后加入含1%(φ)TMCS的BSTFA衍生剂80μL,在70℃条件下反应,衍生1.5 h后冷却至室温。【结论】该方法简单、方便、重复性好。该方法的建立有助于开展更深入的代谢机制相关研究,为农、林领域开展病原微生物代谢组相关研究提供参考。【Objective】The metabolite pretreatment technology was optimized based on GC-MS to establish a rapid,accurate sample preparation protocol for metabolomics analysis in Metarhizium.【Method】Several sample preparation steps,including cell quenching,metabolite extraction,derivatization and detection were optimized,and the stability of this method was also determined.【Result】The quenching effect of 40%cold ethanol was better than that of other quenching solutions,and the recoveries of nucleic acid and protein of Metarhizium were 9.63%and 11.61%respectively.Total 109 metabolites were obtained by cold methanol,more than those by other methods.The longer derivation time was,the more metabolites could be obtained,and 1.5 h was the best.Too high initial temperature of gas chromatography was not conducive to acquisition of metabolites,and 50℃was the best.The optimal sample preparation conditions were as follows:After quenching with 40%cold ethanol,the supernatant was extracted with 2 mL cold methanol.After centrifugation,the supernatant was dried with N2,and then added with 80μL 20 mg/mL methoxylamine hydrochloride pyridine solution.After severe oscillation for 30 s,the supernatant was reacted at 37℃for 90 min,and cooled to room temperature.Then 80μL BSTFA derivatization agent with 1%(φ)TMCS was added,the derivatization reaction continued for 1.5 h at 70℃,and solution cooled to room temperature.【Conclusion】The method is simple,convenient and reproducible,it is conducive to carry out more in depth studies on metabolic mechanisms,and provides references for related studies on metabolic groups of pathogenic microorganisms in agriculture and forestry.

关 键 词：代谢组学 绿僵菌 淬灭 提取 前处理 GC-MS 

分 类 号：S432.1[农业科学—植物病理学]