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환경과학

收集苜蓿根系分泌物以研究邻苯二甲酸二（2-乙基己基）酯对代谢物产生的影响

Published: June 02, 2023
doi:
10.3791/64470
, , ,
1Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science,Chinese Academy of Sciences, 2College of Resources and Environment,University of Chinese Academy of Sciences

Summary

根系分泌物的分泌通常是植物在胁迫条件下的外部解毒策略。该协议描述了如何通过非靶向代谢组学分析 评估 异生素对苜蓿的影响。

Abstract

根系分泌物是植物根系与周围环境之间信息交流和能量传递的主要媒介。根系分泌物分泌物的变化通常是植物在胁迫条件下的外部解毒策略。该协议旨在引入收集苜蓿根系分泌物的一般指南，以研究邻苯二甲酸二（2-乙基己基）酯（DEHP）对代谢物产生的影响。首先，在水培培养实验中在DEHP胁迫下生长苜蓿幼苗。其次，将植物转移到含有50mL灭菌超纯水的离心管中6小时以收集根系分泌物。然后将溶液在真空冷冻干燥机中冷冻干燥。冷冻样品用双（三甲基硅基））三氟乙酰胺（BSTFA）试剂提取并衍生化。随后，使用气相色谱系统与飞行时间质谱仪（GC-TOF-MS）测量衍生化提取物。然后基于生物信息学方法分析获得的代谢物数据。应深入探讨差异代谢产物和显著改变的代谢途径，以揭示DEHP对苜蓿根系分泌物的影响。

Introduction

邻苯二甲酸二（2-乙基己基）酯（DEHP）是一种合成化合物，广泛用于各种塑料和聚合物中作为增塑剂，以提高其可塑性和强度。在过去的几年中，越来越多的研究表明DEHP是一种内分泌干扰物，对人类和其他动物的呼吸，神经和生殖系统有不利影响1，2，3。考虑到其健康风险，美国环境保护署、欧盟和中国环境监测中心均已将DEHP列入重点污染物清单。由于施用了塑料覆盖和有机肥料、废水灌溉和污泥农场的应用，土壤被认为是DEHP在环境中的重要汇4。不出所料，DEHP在农田土壤中普遍存在，其含量甚至高达毫克/公斤干土在中国5，6。DEHP主要通过根部进入植物，并在土壤生态系统中经历不同营养水平的生物放大作用7。因此，近几十年来，人们对植物中DEHP引起的胁迫提出了重大关注。

植物通常容易受到DEHP暴露的影响。已经观察到DEHP胁迫对种子发芽和正常新陈代谢产生不利影响，从而抑制植物生长发育8，9。例如，DEHP可诱导叶肉细胞氧化损伤，降低叶绿素和渗透剂的含量，提高抗氧化酶活性，最终导致食用植物产量和品质下降10，11。然而，以往关于植物对DEHP胁迫响应的研究大多集中在氧化应激和生理生化特性上。与植物代谢相关的相应机制研究较少。根系分泌物是一个通用术语，描述植物根部分泌并释放到环境中的化合物。它们被认为是植物与根际土壤之间的相互作用介质，在支持植物生长发育方面发挥着重要作用12。众所周知，根系分泌物约占所有光合碳的30%-40%13。在污染环境中，根系分泌物通过新陈代谢或外部排斥来提高植物对污染物胁迫的耐受性14。因此，深入了解植物根系分泌物对污染胁迫的反应可能有助于揭示与细胞生物化学和生物现象相关的潜在机制15。

代谢组学技术提供了一种有效的策略，用于同时测量细胞16，17，组织18甚至生物体19的渗出物内的大量小分子代谢物，包括糖，有机酸，氨基酸和脂质。与传统或经典的化学分析方法相比，代谢组学方法大大增加了可检测的代谢物数量20，这有助于以更高通量的方式鉴定代谢物并鉴定关键代谢途径。代谢组学已广泛应用于胁迫环境下的生物反应研究领域，如重金属21、新兴污染物22和纳米颗粒19。这些关于植物的研究大多集中在植物内部组织的代谢变化上，而很少有关于根系分泌物对环境胁迫的响应的报道。因此，本研究的目的是引入收集苜蓿根系分泌物的一般指南，以研究DEHP对代谢物产生的影响。研究结果将为DEHP后续研究植物代谢组学提供方法指导。

Protocol

该协议的目的是提供一个通用管道，从水培培养实验到代谢组学分析，量化DEHP对苜蓿根系分泌物的影响。 1. 水培培养实验 注意：该协议提供了苜蓿水培培养实验的示例，旨在在不同浓度的DEHP的压力下获得苜蓿（Medicago sativa）幼苗。设置三种处理：不添加任何成分的对照，并在营养液中加标1mg kg-1和10 mg kg-1的di（DEHP。根…

Representative Results

在该实验中，根据上述方法收集，提取和分析苜蓿根系分泌物（图1）。设置三个治疗组：对照组，低浓度DEHP（1mg L−1）和高浓度DEHP（10mg L-1）。 对照色谱共检出778个峰，其中314个代谢物可根据质谱鉴定。如图 2所示，这些代谢物根据相对丰度可分为六种类型：碳水化合物（28.6%），酸（15.58%），脂类（13.87%），醇?…

Discussion

该协议提供了有关如何在DEHP胁迫下收集和测量苜蓿根系分泌物以及如何分析代谢组数据的一般指导。需要密切关注该协议中的一些关键步骤。在水培培养实验中，将苜蓿幼苗在装有不同浓度DEHP营养液的玻璃瓶中水培。在整个培养过程中，玻璃瓶应用铝箔覆盖，以防止玻璃瓶光解并确保所有培养溶液中DEHP浓度的均匀性25，26。DEHP 浓度设定为 1 mg L-1 和 10 …

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家自然科学基金（41877139）、国家自然科学基金重大项目（41991335）、国家重点研发计划（2016YFD0800204）、江苏省自然科学基金（编号：BK20161616）、“135”计划、中国科学院前沿计划（ISSASIP1615）的联合支持。

Materials

Adonitol SIGMA ≥99%
Alfalfa seeds Jiangsu Academy of Agricultural Sciences (Nanjing, China)
Analytical balance Sartorius BSA124S-CW
BSTFA REGIS Technologies with 1% TMCS, v/v
Centrifuge Thermo Fisher Scientific Heraeus Fresco17
Chromatographic column Agilent DB-5MS (30 m × 250 μm × 0.25 μm)
Di(2-ethylhexyl) phthalate Dr. Ehrenstorfer
FAMEs Dr. Ehrenstorfer
Gas chromatography(GC) Agilent 7890A
Grinding instrument Shanghai Jingxin Technology Co., Ltd JXFSTPRP-24
Mass spectrometer(MS) LECO PEGASUS HT
Methanol CNW Technologies HPLC
Methoxyaminatio hydrochloride TCI AR
Microcentrifuge tube Eppendorf Eppendorf Quality 1.5 mL
Oven Shanghai Yiheng Scientific Instrument Co., Ltd DHG-9023A
Pyridine Adamas HPLC
R software statistical analysis software (pathway enrichment, topology)
SIMCA16.0.2  statistical analysis software (OPLS-DA etc)
Ultra low temperature freezer Thermo Fisher Scientific Forma 900 series
Ultrasound Shenzhen Fangao Microelectronics Co., Ltd YM-080S
Vacuum dryer Taicang Huamei biochemical instrument factory LNG-T98
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Tags

AlfalfaRoot ExudatesDi(2-ethylhexyl) PhthalateDEHPXenobioticsMetabolomicsMedicago SativaSeed SterilizationGerminationRoot Exudate CollectionMetabolite ProfilingExtractionCentrifugation
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Ren, W., Zhao, R., Teng, Y., Luo, Y. Collection of Alfalfa Root Exudates to Study the Impact of Di(2-ethylhexyl) Phthalate on Metabolite Production. J. Vis. Exp. (196), e64470, doi:10.3791/64470 (2023).
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