संयंत्र कोशिका दीवार पॉलिमर माइक्रोएरे का उपयोग कर के glycan रूपरेखा
Summary
नामक तकनीक<strong> सी</strong> Omprehensive<strong> पुरूष</strong> Icroarray<strongp></strong> संयंत्र सेल दीवार glycans के लक्षण वर्णन के लिए olymer प्रोफाइलिंग (CoMPP) में वर्णित है. इस विधि मोनोक्लोनल एक लघु माइक्रोएरे विश्लेषणात्मक जैविक संदर्भों की एक विस्तृत रेंज में glycan घटना की जांच की अनुमति देता है मंच के साथ परिभाषित glycan epitopes निर्देशित एंटीबॉडी की विशिष्टता को जोड़ती है.
Abstract
Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 & 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.
Protocol
Representative Results
Discussion
CoMPP दिन के एक मामले में सैकड़ों संयंत्र व्युत्पन्न नमूने की glycan संरचना की रूपरेखा के लिए एक तेज और संवेदनशील तरीका है. इस विधि में पहले से ही उपलब्ध जीवाणु या स्तनधारी lectins, रिसेप्टर्स, और 16 एंटीबॉडी जैस…
Declarações
The authors have nothing to disclose.
Acknowledgements
आईईएम के वित्त पोषण के लिए डैनिश अनुसंधान परिषद (एफ़टीपी और FNU) स्वीकार करना चाहते हैं. ERL एक एआरसी डीपी अनुदान के समर्थन को स्वीकार करता है. अटल बिहारी संयंत्र सेल दीवारों अनुदान में उत्कृष्टता के एआरसी सेंटर के समर्थन को स्वीकार करता है.
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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