Glycan profilering af plantecellevægge Polymerer med Microarrays
Summary
En teknik kaldet<strong> C</strong> Omprehensive<strong> M</strong> Icroarray<strong> P</strong> Olymer Profiling (CoMPP) til karakterisering af plantecellevægge glycans er beskrevet. Denne metode kombinerer af monoklonale antistoffer rettet mod definerede glycan-epitoper med en miniature microarray analytisk platform tillader screening af glycan forekomst i en bred vifte af biologiske sammenhænge.
Abstract
Plant cellevægge er komplekse matricer af heterogene glycaner, som spiller en vigtig rolle i fysiologi og udvikling af planter og give de råvarer til menneskelige samfund (f.eks træ, papir, tekstil-og biobrændstof industri) 1,2. At forstå biosyntesen og funktionen af disse komponenter er fortsat udfordrende.
Cellevæg glycans er kemisk og konformationelt forskelligartede på grund af kompleksiteten af deres byggesten, de glycosylrester. Disse danner bindinger ved flere positioner og afviger i ringstruktur, isomer eller anomere konfiguration, og endvidere substitueret er med et array af ikke-sukkerrester. Glycan sammensætning varierer i forskellige celle-og / eller vævstyper eller sub-domæner af en enkelt celle væg 3. Desuden er deres sammensætning også ændret under udvikling 1, eller som svar på miljømæssige signaler 4.
I exces af 2.000 gener har Plantecellekulturer vægge er komplekse matricer af heterogene glycaner blevet forudsagt at være involveret i cellevæg glycan biosyntese og modifikation i Arabidopsis 5. Imidlertid har relativt få af de biosyntetiske gener blevet funktionelt karakteriseret 4,5. Omvendt genetik fremgangsmåder vanskelige, fordi de gener ofte udtrykkes forskelligt, ofte i lave niveauer, mellem celletyper 6. Også, er mutant undersøgelser ofte hindres af gen-redundans eller kompenserende mekanismer, der sikrer passende cellevæg funktion opretholdes 7. Således nye metoder er nødvendige for hurtigt at karakterisere de mange forskellige glycan strukturer og lette funktionelle genomiktilgange at forstå cellevægsbiosyntesen og modifikation.
Monoklonale antistoffer (mAbs) 8,9 er dukket op som et vigtigt redskab til at bestemme glycan struktur og fordeling i planter. Disse anerkender distinct epitoper til stede i større klasser af plantecellevægge glycans, herunder pectiner, xyloglucans, xylaner, mannaner, glucaner og Arabinogalactaner. For nylig deres anvendelse er blevet udvidet til store screening eksperimenter for at bestemme den relative forekomst af glycaner i en bred vifte af plante-og vævstyper samtidigt 9,10,11.
Her præsenterer vi en microarray-baseret glycan screeningsmetode kaldet Omfattende Microarray Polymer Profiling (CoMPP) (figur 1 & 2) 10,11, som giver flere prøver (100 sek) skal screenes ved hjælp af en miniaturiseret microarray platform med reduceret reagens og prøvevolumener. The Spot signalerne på microarray formelt kan kvantificeres for at give semi-kvantitative data om glycan epitop forekomst. Denne fremgangsmåde er velegnet til at spore glycan ændringer i komplekse biologiske systemer 12 og giver et samlet overblik over cellevæggen præparat især når forudgående kendskab of dette er ikke tilgængelig.
Protocol
Representative Results
Discussion
CoMPP er en hurtig og følsom fremgangsmåde til profilering af glycan sammensætning af hundreder plante-afledte prøver i løbet af få dage. Denne fremgangsmåde supplerer de allerede tilgængelige bakterier eller pattedyr glycan array-platforme til high-throughput screening af kulhydrat interaktioner med glycan-bindingsproteiner, såsom lectiner, receptorer og antistoffer 16. Med en stor mangfoldighed af prober er tilgængelige til påvisning af cellevægs-glycans, er det muligt at opnå oplysninger om gl…
Declarações
The authors have nothing to disclose.
Acknowledgements
IEM vil gerne anerkende det danske Forskningsråd (FTP og FNU) til finansiering. ERL anerkender støtte fra en ARC DP tilskud. AB anerkender støtte fra ARC Centre of Excellence i Plant Cell Walls tilskud.
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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