Name: elucidating the metabolism of 2,4-dibromophenol in plants
Uploaded: 2023-02-10T12:25:00
Description: Watch this Scientific Journal Video about Elucidating the Metabolism of 2,4-Dibromophenol in Plants at JoVE.com

本研究得到了国家自然科学基金（21976160）和浙江省公益性技术应用研究项目（LGF21B070006）的支持。

1.胡萝卜愈伤组织的分化
注意：高压灭菌此处使用的所有设备，并在紫外线灭菌的超净工作台中执行所有操作。
<ol><li>通过将均匀的胡萝卜种子（胡萝卜种子）浸入4°C的去离子水中16小时来春化种子。</li>
	<li>用75%乙醇对春化的种子进行表面消毒20分钟，然后在无菌条件下用无菌去离子水冲洗三次。</li>
	<li>进一步用20%H2O2对种子灭菌20分钟?...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Household+dust+as+a+repository+of+chemical+accumulation:+new+insights+from+a+comprehensive+high-resolution+mass+spectrometric+study.">Household dust as a repository of chemical accumulation: new insights from a comprehensive high-resolution mass spectrometric study.</a> Environmental Science &amp; Technology. 52 (5), 2878-2887 (2018).</li><li>Ren, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxylated+PBDEs+and+brominated+phenolic+compounds+in+particulate+matters+emitted+during+recycling+of+waste+printed+circuit+boards+in+a+typical+e-waste+workshop+of+South+China.">Hydroxylated PBDEs and brominated phenolic compounds in particulate matters emitted during recycling of waste printed circuit boards in a typical e-waste workshop of South China.</a> Environmental Pollution. 177, 71-77 (2013).</li><li>de Wit, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+brominated+flame+retardants+in+the+environment.">An overview of brominated flame retardants in the environment.</a> Chemosphere. 46 (5), 583-624 (2002).</li><li>Sun, J. Q., Chen, Q., Qian, Z. X., Zheng, Y., Yu, S. A., Zhang, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+Uptake+and+Metabolism+of+e,4-Dibromophenol+in+Carrot:+In+Vitro+Enzymatic+Direct+Conjugation.">Plant Uptake and Metabolism of e,4-Dibromophenol in Carrot: In Vitro Enzymatic Direct Conjugation.</a> Journal of Agricultural and Food Chemistry. 66 (17), 4328-4335 (2018).</li><li>Chibwe, L., Titaley, I. A., Hoh, E., Simonich, S. L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+framework+for+identifying+toxic+transformation+products+in+complex+environmental+mixtures.">Integrated framework for identifying toxic transformation products in complex environmental mixtures.</a> Environmental Science &amp; Technology Letters. 4 (2), 32-43 (2017).</li><li>Hollender, J., Schymanski, E. L., Singer, H. P., Ferguson, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nontarget+screening+with+high+resolution+mass+spectrometry+in+the+environment:+ready+to+go.">Nontarget screening with high resolution mass spectrometry in the environment: ready to go.</a> Environmental Science &amp; Technology. 51 (20), 11505-11512 (2017).</li><li>Nafisi, M., Fimognari, L., Sakuragi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplays+between+the+cell+wall+and+phytohormones+in+interaction+between+plants+and+necrotrophic+pathogens.">Interplays between the cell wall and phytohormones in interaction between plants and necrotrophic pathogens.</a> Phytochemistry. 112, 63-71 (2015).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+metabolic+pathways+of+2,4,6-tribromophenol+in+rice+plants.">Multiple metabolic pathways of 2,4,6-tribromophenol in rice plants.</a> Environmental Science &amp; Technology. 53 (13), 7473-7482 (2019).</li><li>Hou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+of+tetrabromobisphenol+A+in+pumpkin.">Glycosylation of tetrabromobisphenol A in pumpkin.</a> Environmental Science &amp; Technology. 53 (15), 8805-8812 (2019).</li></ol>

该协议旨在有效识别植物中异生素的生物转化。该协议的关键步骤是植物愈伤组织的培养。最困难的部分是植物愈伤组织的分化和维持，因为植物愈伤组织容易感染并发育成植物组织。因此，重要的是要确保使用的所有设备都经过高压灭菌，并且所有操作都在无菌条件下进行。植物愈伤组织的分化和维持应在黑暗中进行，以避免自养生长和过度发育。此外，补充到MS培养基中的植物激素的剂量和?...

由于人为活动，越来越多的有机污染物被丢弃到环境中1,2，土壤被认为是这些污染物的主要汇3,4。土壤中的污染物可以被植物吸收，并可能通过作物消费直接进入人体，从而沿着食物链转移到更高的营养级生物中，从而导致意外暴露5,6。植物利用不同的途径代谢异生素进行解毒7;阐明异生素的代谢很重要，因为它控制着植物中污染物的实际命运。由于代谢物可以通过叶子（到大气）或根部排泄，因此在暴露的早期阶段确定代谢物提供了测试更多代谢物的可能性8。然而，使用完整植物的研究需要长期实验和复杂的样品制备方案，这些方案可能会受到各种因素的影响。
因此，植物愈伤组织培养是研究植物中异生素代谢的良好选择，因为它们可以大大缩短治疗时间。这些培养物排除了微生物干扰和光化学降解，简化了完整植物的基质效应，标准化了培养条件，并且需要更少的实验工作。植物愈伤组织培养已成功作为三氯生9、壬基酚10 和戊唑醇8 代谢研究的替代方法应用。这些研究表明，愈伤组织培养物中的代谢模式与完整植物中的代谢模式相似。本研究提出了一种无需复杂和耗时的方案即可有效准确地鉴定植物中异生素代谢物的方法。在这里，我们使用植物愈伤组织培养物与高分辨率质谱法相结合，以分析具有低强度信号的代谢物11,12。
为此，将胡萝卜（胡萝卜变种）愈伤组织悬浮液在130rpm和26°C的摇床中暴露于100μg/ L的2,4-二溴苯酚120小时。 选择2,4-二溴苯酚是由于其破坏性的内分泌活性13 和在土壤中广泛存在14。采用高分辨率质谱法提取并分析代谢物。这里提出的协议可以研究植物 中 可以电离的其他类型的有机化合物的代谢。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,4-dichlorophenoxyacetic acid</td>
			<td>WAKO</td>
			<td>1 mg/L</td>
			<td></td>
		</tr>
		<tr>
			<td>20% H2O2</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>10011218-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>4-n-NP, &#62;99%</td>
			<td>Dr. Ehrenstorfer GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-n-NP-d4</td>
			<td>Pointe-Claire</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-benzylaminopurine</td>
			<td>WAKO</td>
			<td>0.5 mg/L</td>
			<td></td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>1269101-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>7890A-5975&#160;gas chromatography</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACQULTY ultra-performance&#160;liquid chromatography</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amber glass vials</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial climate incubator</td>
			<td>Ningbo DongNan Lab Equipment Co.,LTD</td>
			<td>RDN-1000A-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaves</td>
			<td>STIK</td>
			<td>MJ-Series</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 column</td>
			<td>ACQUITY UPLC BEH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DB-5MS capillary column</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>40071190-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze dryer</td>
			<td>SCIENTZ&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-throughput tissue grinder</td>
			<td>SCIENTZ&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MicrOTOF-QII mass spectrometer</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q system</td>
			<td>Millipore</td>
			<td>MS1922801-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog medium</td>
			<td>HOPEBIO</td>
			<td>HB8469-7</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hexane</td>
			<td>Sigma-Aldrich</td>
			<td>H109658-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen blowing instrument&#160;</td>
			<td>AOSHENG</td>
			<td>MD200-2</td>
			<td></td>
		</tr>
		<tr>
			<td>NP isomers, &#62;99%</td>
			<td>Dr. Ehrenstorfer GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oasis HLB cartridges</td>
			<td>Waters</td>
			<td>60 mg/3 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Research plus</td>
			<td>Eppendorf</td>
			<td></td>
			<td>100-1000 &#181;L</td>
		</tr>
		<tr>
			<td>Seeds of Little Finger carrot (Daucus carota var. sativus)&#160;</td>
			<td>Shouguang Seed Industry Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking&#160;Incubators</td>
			<td>Shanghai bluepard instruments Co.,ltd.</td>
			<td>THZ-98AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid phase extractor</td>
			<td>AUTO SCIENCE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound machine</td>
			<td>ZKI</td>
			<td>UC-6</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-sterilized ultra-clean workbench</td>
			<td>AIRTECH</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

elucidating the metabolism of 2,4-dibromophenol in plants

作物可能广泛暴露于有机污染物，因为土壤是污染物丢弃到环境中的主要汇。这通过食用污染物积聚的食物创造了潜在的人类暴露。阐明作物中异生素的吸收和代谢对于评估人类饮食暴露风险至关重要。然而，对于此类实验，使用完整的植物需要长期实验和复杂的样品制备方案，这些方案可能受到各种因素的影响。植物愈伤组织培养结合高分辨率质谱（HRMS）可为准确、省时地鉴定植物异生素代谢物提供解决方案，避免微生物或真菌微环境的干扰，缩短处理时间，简化完整植物的基质效应。2,4-二溴苯酚是一种典型的阻燃剂和内分泌干扰剂，因其在土壤中的广泛存在及其被植物吸收的潜力而被选为模式物质。本文中，从无菌种子中生成植物愈伤组织并暴露于无菌的含2,4-二溴苯酚的培养基中。结果表明，孵育120 h后，在植物愈伤组织中鉴定出8种2,4-二溴苯酚代谢物。这表明2,4-二溴苯酚在植物愈伤组织中迅速代谢。因此，植物愈伤组织培养平台是评价植物异生素吸收代谢的有效方法。

该协议的步骤如图 1所示。按照实验方案，我们将2,4-二溴苯酚处理的胡萝卜愈伤组织提取物的色谱图与对照组进行了比较，发现2,4-二溴苯酚处理中存在八个不同的峰，但在对照中不存在（图2）。这表明在2,4-二溴苯酚处理的胡萝卜愈伤组织中成功检测到2,4-二溴苯酚的8种代谢物（M562，M545，M661，M413，M339，M380，M424和M187）。此外，在2,4-二溴苯酚处理的?...

Watch this Scientific Journal Video about Elucidating the Metabolism of 2,4-Dibromophenol in Plants at JoVE.com

Elucidating the Metabolism of 2,4-Dibromophenol in Plants

本协议描述了一种简单有效的方法来鉴定植物中的2,4-二溴苯酚代谢物。

阐明植物中2,4-二溴苯酚的代谢

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential ...

植物愈伤组织培养为准确、高效、快速地评估植物中异生素的代谢降解提供了解决方案。植物愈伤组织培养可以排除微生物组或真菌干扰和光化学降解，简化完整植物的基质效应，规范栽培条件，缩短处理时间，需要较少的实验工作。本文提出的方法可以深入了解不同有机污染物对作物的吸收行为和代谢机制，有助于环境风险评估。首先，高压灭菌所有设备并在紫外线灭菌的超净工作台中执行所有操作。用75%乙醇对春化的种子表面消毒20分钟。用无菌去离子水冲洗三次，然后再次用20%过氧化氢消毒20分钟。用灭菌的去离子水洗涤种子六次后，通过在含有1%琼脂凝胶的pH 5.8的高压灭菌，无激素的Murashige和Skoog培养基上无菌发芽，并在26摄氏度下孵育16小时光期15天。种子孵育15天后，将幼苗的下胚轴和子叶切成0.5厘米的小块，得到外植体。在含有 15 至 20 毫升补充有 Oxymon 和藻蓝蛋白的高压灭菌 Murashige 和 Skoog 培养基的培养皿中转化外植体，并在 26 摄氏度的黑暗中孵育三到四周以诱导愈伤组织。使用无菌手术刀和镊子，分离由初始外植体形成的约一厘米直径的愈伤组织。为了进行处理，将2,4-二溴苯酚溶解在10毫升无菌液体Murashige和Skoog培养基中。然后将三克分离的胡萝卜愈伤组织加入制备的2,4-二溴苯酚溶液中。按照手稿中所述在空白对照中制备培养基后，将所有烧瓶在黑暗中孵育。为了制备样品，用0.45微米玻璃纤维过滤器过滤，将愈伤组织与2,3-二溴苯酚处理和对照烧瓶小心分离。收集前用超纯水清洗愈伤组织三次。冷冻干燥所有收集的愈伤组织，并用 0.2 赫兹的高通量组织研磨机将 70 克干愈伤组织匀浆三分钟。使用玻璃微量注射器将50微升替代物氘代4-n-纳米苯酚加入均质愈伤组织中并涡旋一分钟。向加标愈伤组织中加入含有等比例甲醇和水的溶液5毫升，超声处理30分钟，以提取2,4-二溴苯酚和代谢物。提取后，将悬浮液在4摄氏度下以8， 000G离心10分钟，并通过移液收集上清液。将提取物以每分钟一毫升的流速通过亲水亲脂性平衡固相萃取或HLB-SPE滤芯。通过将6毫升甲醇通过HLB-SPE柱来稀释分析物。然后将获得的淋洗液在温和的氮气流下浓缩至一毫升，用于仪器分析。为了进行分析，打开色谱柱加热器门。然后通过将色谱柱入口连接到进样阀并将出口连接到质谱仪的入口来安装超高效液相色谱柱。将溶剂管A和B的末端插入相应的溶剂瓶中。按序列号将样品瓶放置在样品盘的相应位置，然后将样品托盘重新插入样品室。在软件窗口中，单击仪器，然后单击进样方法以编辑液相色谱图的条件。选择MS方法并设置质谱分析参数。单击“文件”，然后单击“新建”以创建并命名数据库。加载上面创建的示例程序，方法是选择 MS 文件，然后选择入口文件和注入体积。通过单击“文件并保存”将数据库保存在项目的示例文件夹中。接下来，在主软件窗口中选择运行并启动，然后单击获取样本数据，然后单击开始样本列表中的确定按钮运行以收集数据。要处理数据，请选择目标数据行，然后单击色谱图窗口以查看MS扫描色谱图。在色谱图中，单击显示，然后单击TIC。单击扫描波DS后，添加迹线和确定以获取子质谱扫描。与对照样品相比，2,4-二溴苯酚处理的胡萝卜愈伤组织提取物的色谱图显示存在八种不同的代谢物。此外，色谱图中没有母体2,4-二溴苯酚峰表明在实验条件下胡萝卜愈伤组织中2,4-二溴苯酚的快速代谢。此外，在胡萝卜愈伤组织中孵育的2,4-二溴苯酚通过与葡萄糖和氨基酸直接结合导致代谢物的形成。所有设备以及Murashige和Skoog培养基都应进行高压灭菌，以确保植物愈伤组织的差异化和维护在无菌条件下进行。遵循该程序的组学方法允许创建环境污染物的清晰植物毒性机制图。

Research

JoVE Journal

Environment

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

重组纤维素酶Cel5A从表达<em&gt;里氏木霉</em&gt;在烟草

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

科研

环境科学

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

植物与放射性示踪剂测量矿质营养和毒物的通量

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

高分辨率红外热成像（HRIT）的冰核和冰传播的植物研究中的应用

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

测量除草剂代谢的双子叶杂草价格与切下的叶片分析

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

人类血小板的LC-MS分析作为研究线粒体代谢的平台

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

采用静态磁场农业植物初始增长率提高

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

用静止封闭室维持顶空装置对稻田甲烷和一氧化二氮通量的评价

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

植物冷冻三维红外视频制作协议

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

循环介导等温扩增 (灯管) 法快速识别烟粉虱

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

木本植物的赛莱默水分布,用低温扫描电子显微镜可视化

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...