糖基谱研究植物细胞壁聚合物，用微阵列
Summary
一种称为<strong> C</strong> omprehensive<strong> M</strong> icroarray<strong> P</strong> olymer探查（CoMPP）植物细胞壁聚糖的特性进行说明。该方法结合了与一个微型芯片分析平台允许筛选聚糖发生在一个广泛的范围内的生物上下文定义的聚糖-抗原决定簇特异性的单克隆抗体。
Abstract
植物细胞壁复杂的异构聚糖在生理和植物的发展，并发挥了重要作用，提供原料，为人类社会（ 如木材，造纸，纺织，生物燃料行业）1,2矩阵。然而，这些成分的生物合成和功能的了解仍然充满挑战。
细胞壁聚糖的化学性质和构象不同的复杂的构建块，糖基。这些形式在多个位置之间的联系，并在环结构的不同，同分异构体或端基异构体的配置，此外，被取代的与非糖残基的一个数组。糖基组合物在不同的细胞和/或组织类型或什至单个单元格的壁3的子域变化。此外，它们的组合物1在开发过程中，或在响应于环境线索4也被修改。
在EX制程的2000基因植物细胞壁异构聚糖被预测要参与细胞壁聚糖的生物合成和修改在拟南芥 5是复杂的矩阵。然而，相对较少的生物合成基因功能特点4,5。反向遗传学方法是困难的，因为基因通常差异表达，往往在低的水平，细胞类型之间的6。此外，突变体的研究往往阻碍了的基因冗余或代偿机制，以确保适当的细胞壁功能是保持7。因此，新的方法，需要迅速表征各种不同的聚糖结构和功能基因组学的方法，以方便了解细胞壁的生物合成和修改。
单克隆抗体（mAb）8,9已成为确定在植物中的糖链结构和分布的重要工具。这些识别区INCT抗原表位存在于植物细胞壁聚糖的主要类别，包括的果胶，xyloglucans，木聚糖，甘露聚糖，葡聚糖和阿拉伯半乳聚糖。最近，他们的使用已经扩展到大规模筛选实验，以确定相对丰富的多聚糖广泛的厂房及组织类型，同时9,10,11。
在这里，我们提出了一种基于微阵列聚糖的检查方法，使多个样本的全面的微阵列名聚合物的分析（CoMPP）（ 图1和图2）10,11（100秒）进行筛选，使用小型化的芯片平台，减少了试剂和样品量。在微阵列上的点信号可以正式量化约聚糖抗原表位发生，得到半定量数据。这种方法非常适合于跟踪聚糖变化的复杂的生物系统12，并提供一个全球细胞壁的组合物的概述，特别是当先验知识òF这是不可用。
Protocol
1。组织收集和准备收集100毫克植物组织的鲜重（至少10 mg干重）的至少一式三份的每个组织的利益。以下步骤描述了从植物组织中的细胞壁物质的准备。在存储组织的情况下，不想要的淀粉酶进行提取细胞壁聚合物如先前所述13之前除去。 均质化样品使用Qiagen公司的TissueLyser II，24管适配器集和3毫米的碳化钨珠（30赫兹，2×30秒），用液氮成细粉。或者，如果只有少数样本正…
Representative Results
的相对丰度聚糖在烟草阿拉塔花6个类型的组织（花药的花丝，花粉，子房，花瓣，萼片和耻辱）被确定使用CoMPP。图3A示出了有代表性的微阵列已与mAb JIM5部分（低）的甲基酯化homogalacturonan（HG），果胶多糖14上发生的表位特异的探测。 CDTA提取物中检测到的所有花组织JIM5表位不过是最高的，在花粉和最低的柱头组织（ 图3A和4A）。有趣的是，JIM5表位也检测到在NaOH在花?…
Discussion
分析的聚糖组成的数百植物性样品在短短几天内，CoMPP是一种快速，灵敏的方法。此方法的补充已经可用的细菌或哺乳动物聚糖阵列平台与多糖结合的蛋白质，如外源凝集素，受体，和抗体16的碳水化合物的相互作用的高通量筛选。随着多样性的探头可用于检测细胞壁聚糖，它有可能获得糖链抗原表位属于多糖在植物细胞壁8,9的所有主要类别的详细信息。然而，这些已查明的8,9…
Disclosures
The authors have nothing to disclose.
Acknowledgements
IEM要感谢丹麦研究委员会（FTP和FNU）的资金。 ERL承认ARC DP授予的支持。 AB承认在细胞壁上授予优秀的ARC中心的支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| 3 mm Tungsten Carbide beads | Qiagen | 69997 | |
| Collection microtubes (1.2 mm) | Qiagen | 19560 | 1.5 ml microfuge tubes can also be used |
| Qiagen TissueLyser II | Qiagen | 85300 | |
| 3 mm glass beads | Sigma Aldrich | Z143928 | |
| CDTA | Sigma Aldrich | 34588 | |
| Cadmium oxide | Sigma Aldrich | 202894 | |
| 1,2-diaminoethane | Sigma Aldrich | 03550 | |
| Nitrocellulose membrane (0.22 μm pore size) | GE-water & process technologies | EP2HY00010 | different pore sized membranes are suitable for different pin types |
| Xact II microarrayer robot | Labnext | 001A | the Xact II robot was fitted with a custom 20 x 20 cm ceramic plate to which the nitrocellulose membrane is attached |
| Xtend RM microarray pins | Labnext | 0037-350 | pins must be suitable for spotting on membranes |
| 384 well microtiter plates (polypropylene) | Greiner | 781207 | |
| Anti-glycan monoclonal antibodies | Plant Probes/ CarboSource/Biosupplies |
Websites; PlantProbes (www.plantprobes.net), Carbosource (www.carbosource.net) and Biosupplies (www.biosupplies.com.au). | |
| Anti-Rat IgG (whole molecule) – Peroxidase antibody produced in goat. | Sigma | A9037 | the type of secondary antibodies depends on the primary antibody used (e.g. raised in rat, mouse, goat etc). |
| SIGMAFAST 3,3′-Diaminobenzidine tablets | Sigma | D4293 | the type of developing reagent depends on the secondary antibodies used and the detection method (colourmetric, or chemiluminecent). |
| SuperSignal West Pico Chemiluminescent Substrate | Thermoscientific | 34080 | see above |
| Xplore Image Processing Software | LabNext | 008 | many software types with automatic gridding tools are available to measure pixel value of microarray spots. |
| Plant polysaccharides | Sigma/Megazyme |
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Cite This Article
Moller, I. E., Pettolino, F. A., Hart, C., Lampugnani, E. R., Willats, W. G., Bacic, A. Glycan Profiling of Plant Cell Wall Polymers using Microarrays. J. Vis. Exp. (70), e4238, doi:10.3791/4238 (2012).