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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 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。 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。 <l…
Representative Results
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?…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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)。
Materials
| Acetamiprid (99.8%) | Dr. Ehrenstorfer | 46717 | CAS No: 135410-20-7 |
| Acetonitrile (CAN, 99.9%) | Tedia | AS1122-801 | CAS No: 75-05-8 |
| Agar | Solarbio Science & Technology | A8190 | CAS No: 9002-18-0 |
| Clothianidin (99.8%) | Dr. Ehrenstorfer | 525 | CAS No: 210880-92-5 |
| Dimethoate (98.5%) | Dr. Ehrenstorfer | 109217 | CAS No: 60-51-5 |
| Imidacloprid (99.8%) | Dr. Ehrenstorfer | 91029 | CAS No: 138261-41-3 |
| Imidaclothiz (99.5%) | Toronto Research Chemical | I275000 | CAS No: 105843-36-5 |
| Kinetin (KT, >98.0%) | Solarbio Science & Technology | K8010 | CAS No: 525-79-1 |
| Omethoate (98.5%) | Dr. Ehrenstorfer | 105491 | CAS No: 1113-02-6 |
| Polyvinylpolypyrrolidone (PVPP) | Solarbio Science & Technology | P8070 | CAS No: 25249-54-1 |
| Sucrose | Tocris Bioscience | 5511 | CAS No: 57-50-1 |
| Thiamethoxam (99.8%) | Dr. Ehrenstorfer | 20625 | CAS No: 153719-23-4 |
| Triphenyltetrazolium Chloride (TTC, 98.0%) | Solarbio Science & Technology | T8170 | CAS No: 298-96-4 |
| 2,4-Dichlorophenoxyacetic Acid (2,4-D, >98.0%) | Guangzhou Saiguo Biotech | D8100 | CAS No: 94-75-7 |
| chiral column | Agilent CYCLOSIL-B | 112-6632 | Chromatography column (30 m × 0.25 mm × 0.25 μm) |
| Gas chromatography (GC) | Shimadu | 2010-Plus | Paired with Flame Photometric Detector (FPD) |
| High-performance liquid chromatography (HPLC) | Agilent | 1260 | Paired with Ultraviolet detector (UV) |
| HSS T3 C18 column | Waters | 186003539 | Chromatography column (100 mm × 2.1 mm × 1.8 μm) |
| Ultra-high-performance liquid chromatography (UPLC) | Agilent | 1290-6545 | Tandem quadrupole time-of-flight mass spectrometer (QTOF) |
| Ultra-high-performance liquid chromatography (UPLC) | Thermo Scientific | Ultimate 3000-Q Exactive Focus | Connected to a Orbitrap mass spectrometer |
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Citar este artigo
Jiao, W., Ge, G., Hua, R., Sun, J., Li, Y., Hou, R. Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves. J. Vis. Exp. (148), e59312, doi:10.3791/59312 (2019).