A combinatory approach for analysis of protein sets in barley sieve-tube samples using EDTA-facilitated exudation and aphid stylectomy
A combinatory approach for analysis of protein sets in barley sieve-tube samples using EDTA-facilitated exudation and aphid stylectomy
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DOI：
10.1016/j.jplph.2007.07.023
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发表时间：
2008-01-01
影响因子：
4.3
通讯作者：
van Bel, Aart J. E.
中科院分区：
文献类型：
--
作者：
Gaupels, Frank;Knauer, Torsten;van Bel, Aart J. E.
比较白粉病侵染与未侵染大麦植物中韧皮部蛋白的方法。在一种方法中，通过花柱切除术收集筛管汁液。切开蚜虫管心针并立即用硅油覆盖以防止任何污染或渗出物的改变。这样，每小时最多可获得1 μ L纯韧皮部汁液。有趣的是，病原体感染后，从microcasterized针渗出减少到不到40%的对照植物，这表明白粉病诱导筛管闭塞机制。与费力的花柱切除术相比，使用EDTA促进渗出以防止钙介导的胼胝质形成是快速和容易的，具有大的体积产量。二维（2D）电泳后，从EDTA溶液和管心针渗出物中提取的蛋白质组的数字叠加显示，两种采样技术的一些主要斑点是相同的。然而，EDTA渗出液也含有大量来源不明的污染蛋白质。一个组合的方法可能是最有利的研究中，韧皮部汁液的蛋白质组成的控制和病原体感染的植物之间进行比较。易化渗出可用于通过2D/质谱法进行差异表达蛋白质的消减鉴定，这需要大量的蛋白质。从花柱切除术中获得的纯韧皮部汁液的参考可能有助于通过数字叠加来确认候选点的韧皮部起源。该方法为研究单子叶植物韧皮部蛋白的差异表达提供了新的机会。(C)2007年，Elsevier GmbH。All rights reserved.
methods for comparison of phloem proteins in powdery mildew-infested vs. non-infested Hordeum vulgare plants. In one approach, sieve tube sap was collected by stylectomy. Aphid stylets were cut and immediately covered with silicon oil to prevent any contamination or modification of exudates. In this way, a maximum of 1 mu L pure phloem sap could be obtained per hour. Interestingly, after pathogen infection exudation from microcauterized stylets was reduced to less than 40% of control plants, suggesting that powdery mildew induced sieve tube-occlusion mechanisms. In contrast to the laborious stylectomy, facilitated exudation using EDTA to prevent calcium-mediated callose formation is quick and easy with a large volume yield. After two-dimensional (2D) electrophoresis, a digital overlay of the protein sets extracted from EDTA solutions and stylet exudates showed that some major spots were the same with both sampling techniques. However, EDTA exudates also contained large amounts of contaminative proteins of unknown origin. A combinatory approach may be most favourable for studies in which the protein composition of phloem sap is compared between control and pathogen-infected plants. Facilitated exudation may be applied for subtractive identification of differentially expressed proteins by 2D/mass spectrometry, which requires large amounts of protein. A reference get loaded with pure phloem sap from stylectomy may be useful for confirmation of phloem origin of candidate spots by digital overlay. The method provides a novel opportunity to study differential expression of phloem proteins in monocotyledonous plant species. (C) 2007 Elsevier GmbH. All rights reserved.