阴离子氧化铈纳米粒子催化清除体内植物活性氧的研究
Summary
在这里, 我们提出了一种合成和表征氧化铈纳米粒子 (nanoceria) 的协议, 用于在体内清除活性氧, nanoceria 在植物组织中进行共焦显微成像, 并在体内用共焦显微镜监测 nanoceria ROS 的清除。
Abstract
活性氧 (ROS) 的积累是植物非生物胁迫反应的标志。ROS 在植物中起着双重作用, 在低水平上充当信号分子, 在高水平上破坏分子。在被胁迫的植物中, ROS 的积聚会破坏代谢物、酶、脂类和 DNA, 从而减少植物生长和产量。氧化铈纳米粒子 (nanoceria) 在体内催化清除活性氧的能力为理解和生物工程植物非生物胁迫耐受提供了独特的工具。在这里, 我们提出了一个合成和表征聚 (丙烯酸) 酸涂层 nanoceria (民警) 的协议, 通过叶片渗透与植物界面的纳米微粒, 并监测其分布和 ROS 清除在体内使用共焦显微镜。目前用于操作植物中 ROS 积累的分子工具仅限于模型物种, 需要费力的转化方法。本协议的体内ROS 清除有可能适用于野生类型的植物, 宽叶和叶结构, 如拟南芥。
Introduction
氧化铈纳米粒子 (nanoceria) 广泛应用于生物, 从基础研究到生物工程, 由于其独特的催化活性氧 (ROS) 清除能力1,2,3。Nanoceria 有 ROS 清除能力由于大量表面氧气空缺交替在二氧化状态 (ce3 +和 Ce4 +) 4,5,6。Ce3 +悬垂键有效地清除 ROS, 而纳米晶格菌株通过氧化还原循环反应7促进这些缺陷部位的再生。Nanoceria 最近也被用于研究和工程植物功能8,9。非生物胁迫下的植物积累了 ROS, 对脂质、蛋白质和 DNA 造成氧化损伤10。在 nanoceria 植物中, 在体内活性氧的催化清除能在高光、高温和冷应力下改善植物光合作用8。nanoceria 对土壤的施用也增加了小麦的生物质量和籽粒产量11;nanoceria 处理的油菜 (甘蓝) 植株在盐胁迫下具有较高的植物生物量12。
Nanoceria 提供生物工程师和植物生物学家一个基于纳米技术的工具, 以了解非生物胁迫反应和提高植物非生物胁迫耐受性。Nanoceria 的体内活性氧清除能力是独立于植物物种, 并在植物组织的简便交付有潜力, 使广泛应用外的模型有机体。不像其他基于基因的方法, nanoceria 不需要产生植物线与抗氧化酶的过度表达, 以提高 ROS 清除能力13。nanoceria 对植物的叶片浸润是实验室研究的一种实用方法。
本议定书的总目标是描述 1) 带负电荷的聚 (丙烯酸) 酸 nanoceria (民警) 的合成和表征, 2) 在整个叶细胞内提供和跟踪全国民警, 3) 监测在体内。本协议合成了带负电荷的聚丙烯酸 (nanoceria) 酸 (民警), 并以其吸收谱、流体力学直径和泽塔电位为特征。我们描述了一种简单的叶片入渗方法, 以提供民警到植物叶组织。在体外显像的纳米颗粒在叶肉细胞中的分布, 用荧光染料 (DiI) 标记民警 (DiI), 并通过共焦荧光显微镜观察纳米粒子。最后, 我们解释如何通过共聚焦显微镜监测体内的ROS 清除。
Protocol
1. 种植芥的植物 在5厘米 x 5 厘米的一次性花盆中播种一个.把32这些花盆放入一个装满水的塑料托盘 (0.5 厘米深), 把塑料托盘和植物转移到植物生长室。 设置生长室设置如下: 200 µmol/ms 光合活性辐射 (PAR), 24 1 °c 日和 21 @ 1 °c 夜, 60% 湿度, 14/10 h 天/夜光政权分别。 在一周的发芽之后, 每壶都要细细地留下一株。注意保持每个花盆中大小相同的幼苗。 <…
Representative Results
民警的合成和表征.民警的合成、纯化和特点遵循了议定书第2节所述方法。图 1A显示了硝酸铈、临机局、硝酸铈和临机局的混合物和民警的溶液的着色。在合成了民警后, 从白色到浅黄色的颜色变化是可见的。用 10 kDa 过滤器纯化后, 民警的特征是紫外-可见光分光光度计。在 271 nm (图 1</st…
Discussion
在本协议中, 我们描述了在植物叶肉细胞内的纳米粒子的合成、表征、荧光染料标记和共焦成像, 以展示其体内活性氧清除活性。从硝酸铈的混合物和氢氧化铵中的临机溶液中合成了民警。民警的特点是吸收 spectrophotomery 和浓度确定使用啤酒 Lamberts 法。泽塔电位测量证实了民警的负电荷表面, 以增强对叶绿体8的传递。用荧光 DiI 染料对民警进行标记,在叶片叶肉细胞?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了加利福尼亚大学、河畔分校和美国农业部食品与农业研究所的支持, 孵化项目 1009710 J.P.G.。该材料的基础是国家科学基金会根据1817363号赠款资助的 J.P.G. 的工作。
Materials
| Cerium (III) nitrate hexahydrate | Sigma-Aldrich | 238538-100G | |
| Molecular Biology Grade Water, Corning | VWR | 45001-044 | |
| Falcon 50 mL Conical Centrifuge Tubes | VWR | 14-959-49A | |
| Poly (acrylic acid) 1,800 Mw | Sigma-Aldrich | 323667-100G | |
| Fisherbrand Digital Vortex Mixer | Fisher Scientific | 02-215-370 | |
| Fisherbrand Digital Vortex Mixer Accessory, Insert Retainer | Fisher Scientific | 02-215-391 | |
| Fisherbrand Digital Vortex Mixer Accessories: Foam Insert Set | Fisher Scientific | 02-215-395 | |
| Ammonium hydroxide solution | Sigma-Aldrich | 05002-1L | |
| PYREX Griffin Beakers, Graduated, Corning | VWR | 13912-149 | |
| RCT basic | IKA | 3810001 | |
| Eppendorf Microcentrifuge 5424 | VWR | 80094-126 | |
| Amicon Ultra-15 Centrifugal Filter Units | Millipore-Sigma | UFC901024 | |
| Allegra X-30 Series Benchtop Centrifuge | Beckman Coulter | B06314 | |
| UV-2600 Sptecrophotometer | Shimadzu | UV-2600 120V | |
| Whatman Anotop 10 syringe filter | Sigma-Aldrich | WHA68091102 | |
| BD Disposable Syringes with Luer-Lok Tips | Fisher Scientific | 14-829-45 | |
| Zetasizer Nano S | Malvern Panalytical | Zen 1600 | |
| 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate | Sigma-Aldrich | 42364-100MG | |
| Dimethyl Sulfoxide, ACS | VWR | BDH1115-1LP | |
| Sunshine Mix #1 LC1 | Green Island Distributors, Inc | 5212601.CFL080P | |
| Adaptis 1000 | Conviron | A1000 | |
| TES, >99% (titration | Sigma-Aldrich | T1375-100G | |
| Magnesium chloride | Sigma-Aldrich | M8266-1KG | |
| Air-Tite All-Plastic Norm-Ject Syringe | Fisher Scientific | 14-817-25 | |
| Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers | Fisher Scientific | 06-666A | |
| Carolina Observation Gel | Carolina | 132700 | |
| Corning microscope slides, frosted one side, one end | Sigma-Aldrich | CLS294875X25-72EA | |
| Cork Borer Sets with Handles | Fisher Scientific | S50166A | |
| Perfluorodecalin | Sigma-Aldrich | P9900-25G | |
| Micro Cover Glasses, Square, No. 1 | VWR | 48366-045 | |
| Leica Laser Scanning Confocal Microscope TCS SP5 | Leica Microsystems | TCS SP5 | |
| 2′,7′-Dichlorofluorescin diacetate | Sigma-Aldrich | D6883-250MG | |
| Dihydroethidium | Sigma-Aldrich | D7008-10MG | |
| Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL | Fisher Scientific | 05-408-129 | |
| Eppendorf Uvette cuvettes | Sigma-Aldrich | Z605050-80EA | |
| Chlorophyll meter | Konica Minolta | SPAD-502 |
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Cite This Article
Newkirk, G. M., Wu, H., Santana, I., Giraldo, J. P. Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles. J. Vis. Exp. (138), e58373, doi:10.3791/58373 (2018).