Name: study on the metabolism of six systemic insecticides in a newly established cell suspension culture derived from tea (camellia sinensis l.) leaves
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这项工作得到了国家重点研究与发展计划(2016YFD0200900)、国家自然科学基金(第31772076号、第31270728号)、中国博士后科学基金会(2018M630700)和开放基金的支持。茶植物生物学与利用国家重点实验室(SKLTOF20180111)。

1. 茶文化
注:无菌叶来自在研究组17中首次开发的体外生长的植物系。除孵化器中的培养时间外,所有程序均在第5节进行无菌层流罩。
<ol><li>在高压灭菌之前,将两种介质的pH值(Murashige和Skoog [MS] 基底介质和甘博格的B5液体介质)调整到5.8。C,20分钟。</li>
	<li>用剪刀沿着无菌叶的中间静脉切开,然后将每半部分细分为约 0.3 厘米 x 0.3 厘米的培养皿中的小块。</li>
	<l...

<ol>
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本文介绍了在茶植物组织中建立农药代谢模型的详细过程,包括植物外植物的选择、细胞活力的测定、茶细胞悬浮培养的建立等。活动。植物组织的任何部分都可以用来在消毒环境中启动callus25。在这项研究中,茶叶被选择用于开叶,不仅因为叶子比地下部分受到的污染要小,还因为它们是作物的可食用部分和杀虫剂应用的主要目标。
在这项研究中,比较了从田间?...

茶是世界上消费最广泛的非酒精饮料之一。茶是由木本多年生山茶的叶子和芽,茶植物生长在广阔的种植园,易受大量害虫3,4。有机磷和尼古丁类杀虫剂常被用作系统性杀虫剂5,以保护茶树免受害虫,如白蝇,叶漏斗,和一些白鳍豚物种6,7。施用后,这些杀虫剂被吸收或转移到植物中。在植物内部,这些系统杀虫剂可以通过水解、氧化或植物酶的还原反应转化。这些转化产物可能比母化合物更极性,毒性更低。然而,对于一些有机磷酸盐,一些产品的生物活性较高。例如,乙酰胺被代谢成毒性更大的甲胺磷8,9,和二甲苯酸盐成欧美磷10,11。因此,植物代谢研究对于确定植物中农药的命运非常重要。
植物组织培养物已被证明是研究农药代谢的有用平台,其识别代谢物与在完整植物13、14、15中发现的代谢物相似。使用组织培养物,特别是细胞悬浮培养物,有几个优点。首先,可以进行无微生物的实验,从而避免微生物对农药转化或降解的干扰。其次,组织培养提供一致的材料,随时使用。第三,代谢物比从完整植物中提取更容易,组织培养物通常具有较少的相互作用化合物和较低的化合物复杂性。最后,组织培养可以更容易地用于比较一系列农药代谢在一个单一的实验16。
在这项研究中,成功地建立了从无菌生长的茶树叶中提取的细胞悬浮液。然后用茶细胞悬浮培养法比较六种系统杀虫剂的耗散行为。
这个详细的协议旨在提供一些指导,以便研究人员可以建立一个植物组织培养平台,有用的研究异种生物在茶中的代谢命运。

<table border="0" cellpadding="0" cellspacing="0" style="width:674px;" width="673">
	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Acetamiprid (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">46717</td>
			<td style="width:123px;">CAS No: 135410-20-7</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Acetonitrile (CAN, 99.9%)</td>
			<td style="width:188px;">Tedia</td>
			<td style="width:175px;">AS1122-801</td>
			<td style="width:123px;">CAS No: 75-05-8</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Agar</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">A8190</td>
			<td style="width:123px;">CAS No: 9002-18-0</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Clothianidin (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">525</td>
			<td style="width:123px;">CAS No: 210880-92-5</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Dimethoate (98.5%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">109217</td>
			<td style="width:123px;">CAS No: 60-51-5</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Imidacloprid (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">91029</td>
			<td style="width:123px;">CAS No: 138261-41-3</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Imidaclothiz (99.5%)</td>
			<td style="width:188px;">Toronto Research Chemical</td>
			<td style="width:175px;">I275000</td>
			<td style="width:123px;">CAS No: 105843-36-5</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Kinetin (KT, &#62;98.0%)</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">K8010</td>
			<td style="width:123px;">CAS No: 525-79-1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Omethoate (98.5%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">105491</td>
			<td style="width:123px;">CAS No: 1113-02-6</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Polyvinylpolypyrrolidone (PVPP)</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">P8070</td>
			<td style="width:123px;">CAS No: 25249-54-1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Sucrose</td>
			<td style="width:188px;">Tocris Bioscience</td>
			<td style="width:175px;">5511</td>
			<td style="width:123px;">CAS No: 57-50-1</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Thiamethoxam (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">20625</td>
			<td style="width:123px;">CAS No: 153719-23-4</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Triphenyltetrazolium Chloride (TTC, 98.0%)</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">T8170</td>
			<td style="width:123px;">CAS No: 298-96-4</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:188px;">2,4-Dichlorophenoxyacetic Acid (2,4-D, &#62;98.0%)</td>
			<td style="width:188px;">Guangzhou Saiguo Biotech</td>
			<td style="width:175px;">D8100</td>
			<td style="width:123px;">CAS No: 94-75-7</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:77px;width:188px;">chiral column</td>
			<td style="width:188px;">Agilent CYCLOSIL-B</td>
			<td>112-6632</td>
			<td style="width:123px;">Chromatography column (30 m &#215; 0.25 mm &#215; 0.25 &#956;m)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">Gas chromatography (GC)</td>
			<td style="width:188px;">Shimadu</td>
			<td>2010-Plus</td>
			<td style="width:123px;">Paired with Flame Photometric Detector (FPD)&#160;&#160;</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">High-performance liquid chromatography (HPLC)</td>
			<td>Agilent</td>
			<td>1260</td>
			<td style="width:123px;">Paired with Ultraviolet detector (UV)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">HSS T3 C18 column</td>
			<td style="width:188px;">Waters</td>
			<td>186003539</td>
			<td style="width:123px;">Chromatography column (100 mm &#215; 2.1 mm &#215; 1.8 &#956;m)</td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:188px;">Ultra-high-performance liquid chromatography (UPLC)</td>
			<td style="width:188px;">Agilent</td>
			<td>1290-6545</td>
			<td style="width:123px;">Tandem quadrupole time-of-flight mass spectrometer (QTOF)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">Ultra-high-performance liquid chromatography (UPLC)</td>
			<td style="width:188px;">Thermo Scientific</td>
			<td>Ultimate 3000-Q Exactive Focus</td>
			<td style="width:123px;">Connected to a Orbitrap mass spectrometer</td>
		</tr>
	</tbody>
</table>

study on the metabolism of six systemic insecticides in a newly established cell suspension culture derived from tea (camellia sinensis l.) leaves

利用茶植物体外组织研究杀虫剂代谢的平台。从无菌茶树叶诱导形成松散的卡苏在Murashige和Skoog(MS)基底培养基与植物激素2,4二氯苯氧乙酸(2,4-D,1.0毫克L-1)和基尼汀(KT,0.1毫克L-1)。在3或4轮次培养后形成,每轮持续28天。然后,将松弛的卡鲁斯(约3克)接种到含有相同植物激素的B5液体介质中,并在25~1°C的黑暗中,在摇动的培养箱(120 rpm)中培养。在3⁄4亚培养后,以1:1和1:2(悬浮母液:新鲜介质)的亚培养比建立从茶叶中提取的细胞悬浮液。利用该平台,在茶叶衍生细胞悬浮培养中加入六种杀虫剂(每片甲氨酰胺、伊米达洛狄德、乙酰胺、伊米达布蒂、二甲苯酸盐和奥米霍特)。利用液相色谱和气相色谱跟踪杀虫剂的代谢。为了验证茶细胞悬浮物培养物的效用,使用质谱法比较了处理过的细胞培养和完整植物中存在的三甲氧西和二甲苯甲酸酯的代谢物。在治疗茶细胞培养中,发现七种三甲氧烷代谢物和两种二甲苯醚代谢物,而在治疗完好的植物中,只发现两种三甲氧烷代谢物和一种二甲苯甲酸酯。与使用完整的茶树相比,使用细胞悬浮液简化了代谢分析,尤其是对于茶等困难的基质。

在MS介质上种植28天后,通过测量污染、褐化和诱导,从田间种植的茶树和从在无菌环境中在体外生长的茶树中切去的叶子中,对叶的诱导进行了比较。图 1A.在20、37、62和90天的文化上,卡鲁斯的增长被记录下来(图1B)。在整整90天的栽培过程中,从体外生长的叶子中提取的卡鲁斯比从田间生长的叶子中提取的卡鲁斯更活跃。无菌叶子的?...

Watch this Scientific Journal Video about Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves at JoVE.com

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

这项工作提出了建立从茶(山茶)叶衍生的细胞悬浮培养物的协议,可用于研究整个植物可以采用的外部化合物的代谢,如杀虫剂。

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

茶是全世界最受欢迎的饮料之一。杀虫剂有时被用来保护茶树免受害虫的侵害。茶树中的酶可以为此制造一些系统杀虫剂。然后通过氧化、水解或还原反应进行整合。植物细胞悬浮培养物可作为一种，容易研究植物代谢的农药行为。此方法有几个优点。首先，可以在自由微生物条件下操作，从而避免微生物对农药降解的干扰。其次，它随时可以为我们提供恒定的材料。最后，在单个实验中也可以很容易地比较农药行为理论。本研究从茶叶的可变颜色中建立茶细胞悬浮培养物。然后利用细胞悬浮液的最佳培养状态比较六种农药的耗散行为。这个实验将由焦伟庭和葛国勤进行。这是我们的茶叶无菌植物，作为外植的来源。用剪刀沿着无菌叶子的中间翼切。然后，在培养皿中，将每半部分细分成约0.3倍0.3厘米的小片。将无菌外植植物放在含有 2、4-D 和 KT 的 MS 视觉介质上。六种外植剂可以放在一个300毫升的烧瓶中。在黑暗中25摄氏度的恒定温度下培养叶子。28天后，选择第一代诱导卡卢斯，然后转移到新鲜的烧瓶。在三到四个亚文化之后获得松散和可变的调用。在无菌条件下，使用无菌手术刀片将固体介质中的可变和松散的卡卢斯切成小块。我们大约三克的卡卢斯小片。将调用放入包含 2、4-D 和 KT 的 B5 中。在黑暗中摇动的培养箱中，在恒定温度下培养液体细胞悬浮液。经过七到十天的栽培，取出文化烧瓶，让它们站立几分钟。以上流剂作为这里的沉积物将迫使培养到新鲜的介质。拆下沉淀的大型调用。在四到三个亚培养周期（每个28天）后，获得最终精心梳理的细胞悬浮培养。将活细胞样本在一百摄氏度下保持十分钟，作为对照细胞，然后进行活体染色。在6000G下将所有细胞悬浮培养物离心8分钟，取出上流液，然后将细胞悬浮在5毫升PBS缓冲液中，然后用手摇动1分钟。加入 2.5 毫升的 TTC 溶液，然后再次握手。在13摄氏度的站立培养箱中孵育混合物一小时。添加四百微升的等分，对所有新烟碱硫胺素、乙酰胺、伊米达克洛普里德和伊米德普罗西酶的库存溶液进行灭菌。全部到有机第一阶段。二甲酸酯和甲基酸盐分别进入细胞悬浮培养。在恒定温度下用杀虫剂培养细胞悬浮液样本，并摇动孵化器速度。在不同的日子采集样品。要测试含有新烟碱的样品，请去除同质细胞培养的1毫升等同。放入1.5毫升塑料离心管中，在4000G下离心两分钟。通过 HPLC-UV 和 UPLC QTOF 分析之前，通过 0.22 微米滤膜传递上清液。要测试样品，请继续有机第一阶段。取出细胞培养的500微升等分，放入35毫升离心管或1.5毫升塑料离心管中。将 0.1 克氯化钠和 5 毫升溶剂萃取到 500 微升样品的 35 毫升离心管中。漩涡混合物一分钟。然后让他们休息10分钟。将 2.5 毫升上充液放入 10 毫升玻璃管中，在 14 摄氏度下使用氮蒸发器蒸发至近乎干燥。用一毫升丙酮溶解残留物。在GC-FPD分析之前，Vortex通过0.22微米滤膜进行一分钟。在四升塑料锅中放入五株植物十五天。将每毫升零微克或每毫升100微克的硫胺酮或二甲基苯甲苯分别加入到塑料罐中。根据前一种方法准备完整的植物样品，除了预浸泡，然后通过轨道rap质谱法进行分析。使用 HPLCUV 检测 254 纳米波长的硫胺素和乙酰胺的含量和代谢产物，以及波长为 270 纳米的 Imidacloprid 和 Imideproxase 的含量和代谢产物。使用管柱通过 GC-FPD 检测二甲酸酯和甲基酸盐的含量。使用带碳-18柱的 UPRC QTOF 检测细胞培养中的杀虫剂代谢物。使用 UPLC Orbitrap 质谱仪检测试验植物提取物中的杀虫剂代谢物。一种无菌植物的卡卢斯具有较低的污染棕色，但更高的电感性比从采摘的茶叶的卡卢斯。从无菌叶子中提取的卡卢斯总是亮黄色，而从采摘的叶子中提取的卡卢斯是白色，有棕色斑点。当 KT 浓度为每升 0.1 毫克时，卡卢斯的颜色发黄，质地松散。当2，4-D的浓度是每升1毫克，KT的浓度为每升0.5毫克时，卡卢斯的生长速度最高，高达61.5%。卡卢斯的生长数据最好，卡卢斯的生长率最高达到46.9%，因此，植物家的最佳组合是每升1毫克2，4-D和0.1毫克每升KT的茶卡卢斯生长。当亚文化周期为28天时，树种完全覆盖了第四亚文化的叶子。卡卢斯已经大力生长与黄色和松散的质地，没有棕色。在比较了卡卢斯的毛数据和细胞悬浮液的颜色后，B5液体介质更适合细胞悬浮培养。细胞悬浮培养的OD值用于表示细胞生长量。在75天的栽培中，每40毫升15克的消耗臭氧值明显高于其他两个比率。通过TTC染色测试了茶细胞悬浮培养中的细胞生存能力。无色TTC化合物可以通过活细胞线粒体的脱氢酶转化为红蛋白，但死细胞的颜色不能改变。这是建立茶细胞悬浮培养的整个过程。这是首次采用细胞悬浮培养技术对茶叶中不同农药代谢的比较研究。发现了几种农药代谢物，其中一些在保存的茶厂中进一步研究。这种新方法可以作为茶叶或其他植物农药代谢研究的模型。

Research

JoVE Journal

Biochemistry

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

映射代谢: 直接在组织中监测乳酸脱氢酶活性

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

高速离心条件下 u937 细胞的细胞分裂

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...