Bestimmung des Phytatgehalts in Samen durch HPIC-Analyse .1 Extraktion der Inositolphosphate aus Samen

Der Samen wurde zur Bestimmung des Inositolphosphatgehaltes mit einer Kugelmühle homogenisiert und das Mehl anschließend bei 65 °C für 48 h getrocknet. 50 mg des

Sa-menmehls wurden mit 1 ml 0,4 M HCl versetzt und gründlich durch vortexen resuspendiert. Die Probe wurden über Nacht bei Raumtemperatur mit 200 rpm geschüttelt und anschließend die unlöslichen Bestandteile durch 30minütige Zentrifugation in einer Tischzentrifuge mit 13000 rpm abgetrennt. Der Überstand wurde für die HPIC-Analyse verwendet.

5.4.8.6.2 HPIC-Analyse

Je 0,5ml jeder Probe sowie der Nullprobe wurde nach Zentrifugation des Überstandes in HPLC-Probenfläschen (Agilent) überführt und die Bestimmung mittels HPLC (Chromatography Compartment AS50 Dionex) bei 25 °C durchgeführt. Die Detektion der Inositolphosphate erfolgte durch eine der HPLC-Säule nachgeschalteten Säulenderivatisierung. Mittels Autosampler (AS50 Dionex) wurden jeweils 90 µl der Pro-ben auf eine Anionaustauschersäule (Mono-Q-5/50 GL Säule (Amersham) mit einer Fluss-rate von 1 ml/min aufgetragen. Die unterschiedlich phosphorylierten Inositolphosphate wurden durch einen Salzsäuregradienten (Gradientenpumpe GP40 (Dionex)) von der Säule eluiert und dann im Verhältnis 2:1 mit Eisen(III)-Nitrat-Perchlorsäure-Lösung (1 g Fe(N03)3 in 1000 ml 0,33 M HClO4) gemischt (post column reactor (Dionex). Der Salzsäuregradient wurde mittels 0,005 M und 0,5 M Lösungen erzeugt. Dabei wurde in-nerhalb von 20 min die HCl-Konzentration bis auf 0,4 M in einem linearen Gradienten erhöht. Die Eisen(III)-nitrat-Perchlosäure-Lösung wurde mit 0,5 ml/min unter Verwendung einer isokratischen Pumpe (IP20 Dionex) zum Eluat zugesetzt. Die Detektion (UV-Detektor AD25 (Dionex)) des derivatisierten Eluates erfolgte bei 290 nm. Die Daten wur-den anschließend mit der Software Peaknet 6.3 (Dionex) und Chromeleon 6.7 (Dionex) ausgewertet und dokumentiert. Die Kalibrierung zur Bestimmung der Konzentration des Inositolhexakisphosphates wurde auf Grundlage der Analysen des Institutes für Biochemie und Molekularbiolgie des Universitätsklinikums Hamburg-Eppendorf durchgeführt und im Anhang unter 7.4 aufgeführt. Das HPIC-Programm wurde im Anhang unter 7.3 aufge-schrieben.

5.4.8.7 Wurzelmorphologie

Die Analyse der Wurzel erfolgte mit Hilfe des Programms WinRhizo Pro 2005b (Regent Instruments Inc.).

5.4.8.7.1 Spezifische Wurzellänge

Zur Bestimmung der spezifischen Wurzellänge wurde die gesamte frische Wurzel in 1 cm lange Stücke geschnitten. Die Wurzelstücken wurden durchmischt und eine 500 mg Unter-probe gleichmäßig in einer mit destilliertem H2O gefüllten Plexiglaswanne verteilt. Der Scan der Wurzelstücken erfolgte mit dem Durchlichtscanner Epson Expression 1680 Mo-dell EU-35. Die Auswertung fand mit dem Programm WinRhizo Pro 2005b statt. Bei der Berechnung wurde auf die Trockenmasse der Wurzel bezogen (Meter/g Trockenmasse).

5.4.8.7.2 Gesamtwurzellänge

Die Gesamtwurzellänge wurde mit dem Programm WinRhizo Pro 2005b (Regent Instru-ments Inc.) bestimmt. Sie errechnet sich aus dem Produkt von gemessener Wurzellänge der Unterprobe und Frischmasse der Gesamtwurzel geteilt durch die Frischmasse der Unter-probe.

5.4.8.7.3 Durchschnittlicher Wurzeldurchmesser

Der durchschnittliche Wurzeldurchmesser wurde ebenfalls mit dem Programm WinRhizo Pro 2005b (Regent Instruments Inc.) bestimmt. Er ergibt sich aus dem Quotienten aus Flä-cheninhalt der Wurzel und der gesamten Wurzellänge.

5.5 Software

Der Text und die Tabellen wurden mit MS-Word2007 verfasst. Die Diagramme wurden mit MS-Excel2007 erstellt. Die Bildbearbeitung erfolgte mit Corel Graphics Essentials. Die digitale Bilddokumentation fand mit Syngene von Merck statt. Die Auswertung der pho-tometrischen Messung erfolgte mit Ascent Software Version 2.6 von Thermo Scientific.

Die Sequenzen wurde mit Vector NTI Version 9 von Invitrogen ausgewertet. Die Statstikanalysen wurden mit PASW Statistics 18 durchgeführt.

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Im Dokument Expression bakterieller Phytasen in Pflanzen (Seite 161-179)