植物多聚核糖体剖面的简单方法
Summary
This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.
Abstract
Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.
Introduction
蛋白质的合成是在所有单元电池1的基本和大力昂贵的过程。首先，细胞必须在生产翻译机器中，核糖体的投资能量。例如，一个活跃分裂的酵母细胞产生每分钟高达2000核糖体。这样的生产需要高达总转录活性的60％，到小区2的总剪接活性的90％。此外，需要能量的氨基酸，氨酰tRNA和肽键的合成。在植物中，从三磷酸腺苷3 4.5至5.9分子添加一个氨基酸的肽链的成本。因此，它是不奇怪的mRNA的蛋白质的翻译调控的主要部位，特别是当它涉及到处理变化的环境条件。
翻译起始的一步，那就是与一个核糖体mRNA的关联，是调控的主要对象翻译4。作为翻译的调节的结果，以及其他转录后调控的步骤中，只有40％的蛋白质浓度的变化可通过mRNA的丰度5,6进行说明。因此，总mRNA的研究给出了关于蛋白丰度比较差的信息。另一方面，mRNA的核糖体与关联给出更好地了解蛋白质丰度通过给访问那些参与翻译的mRNA。积极翻译的mRNA与核糖体的几个称为多聚核糖体的结构有关。相反，翻译不好的mRNA将只有一个核糖体（monosome）相关联。因此，一个mRNA的转译状态可以通过监视与核糖体7的关联进行评估。
这个协议描述从六个日龄拟南芥幼苗，RNA的随后分离多聚核糖体的分离，结果的分析。宝lysomes和monosomes通过蔗糖密度梯度分离。梯度被收集成六个馏分。一些级分的汇集，以获得三个阱分离的部分：多核糖体，monosomes和轻馏分（以下称为上清液），其中包含不与核糖体相关的自由60S和40S核糖体亚基和的mRNA。全局翻译活性可以通过产生一个多核糖/ monosome比率，这是由该曲线下的面积的积分确定来估计，并通过比较多聚核糖体轮廓。的mRNA和蛋白质，然后从不同的级分提取并用于通过RT-PCR，定量RT-PCR，Northern印迹，微阵列，蛋白质印迹或蛋白质组学分析。该协议已经过验证的其他植物和组织。
执行此协议所需的设备在大多数实验室通常发现：没有必要为一个梯度壶。添加下一个防止冻结之前每层■从层的任意组合或干扰。无管穿孔被用于其可以通过在梯度的玻璃毛细管的浸没来实现梯度集合。因此，昂贵的超速离心管保持完好，并且可以重复使用很多次。总的来说，这使得本协议核糖体分析一个简单而廉价的方法。
Protocol
Representative Results
Discussion
The protocol we present here is an easy and cheap method for generating polysome profiles and isolating mRNAs associated with polysomes, single ribosomes or free of ribosomes. A wide range of different polysome fractionation methods is described in the literature. The method we have described here has been optimized to keep only the necessary compounds and has been adapted for plant material. In particular, we reduced the amount of detergent11 and added chloramphenicol to the buffer to fix the chloroplastic ri…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由法国国家研究署（ANR-14-CE02-0010）的支持。我们感谢本杰明博士场和的Elodie Lanet博士的手稿的批判性阅读。我们感谢米歇尔Terese先生的视频编辑的帮助。
Materials
| Ultracentrifuge tube, thinwall, polyallomer – 13.2 ml | Beckman Coulter | 331372 | |
| Ultracentrifuge tube, thinwall, polyallomer – 38.5 ml | Beckman Coulter | 326823 | |
| Glass capillary tube | Drummond Scientific | 1-000-1000 | |
| Ultracentrifuge | Beckman Coulter | Optima series | |
| Ultracentrifuge Rotor SW41 | Beckman Coulter | 331362 | |
| Ultracentrifuge Rotor SW32 | Beckman Coulter | 369650 | |
| Peristaltic pump | Any | ||
| Tygon R3607 polyvinyl chloride tubing | Fisher Scientific | 070534-22 | Polyvinyl chloride tubing, 2.29 mm |
| Fraction collector Model 2110 | Bio-Rad | 731-8120 | |
| UV cuvette | Hellma | 170.700-QS | Quartz flow-through cuvette |
| UV Spectrophotometer | Varian | Cary50 | Read every 0.0125 sec |
| All chemicals | Any | Use only Molecular Biology Grade | |
| Murashige and Skoog Basal Salt Mixture (MS) | Sigma-Aldrich | M5524 | |
| Rnase-Free water | Any | ||
| Petri Dishes | Fisher Scientific | 10083251 | |
| Octylphenoxy poly(ethyleneoxy)ethanol, branched (Nonidet P40) | Euromedex | UN3500 | |
| Linear acrylamide (acryl carrier) | ThermoFischer scientific | AM9520 | RNA precipitation carrier |
| OriginPro 8 | OriginLab | Analysis software |
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