Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap.
Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap.
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DOI：
10.1093/jxb/ern181
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发表时间：
2008
影响因子：
6.9
通讯作者：
Kehr J
中科院分区：
文献类型：
--
作者：
Gaupels F;Buhtz A;Knauer T;Deshmukh S;Waller F;van Bel AJ;Kogel KH;Kehr J
筛管不仅是光合产物的运输管道，而且也是大分子和其他参与筛管维持和系统信号传导的化合物的运输管道。为了获得足够量的纯韧皮部分泌物从大麦植物的蛋白质和mRNA组成的分析，先前描述的花柱切除设置进行了优化。将蚜虫置于密封笼中，在探针微烧灼后立即用水饱和的硅油淹没。用连接到泵的毛细管收集渗出的韧皮部汁液。平行使用多达30株植物和600只蚜虫（禾谷缢管蚜），平均10 μl的韧皮部汁液可以在6小时内取样。在第一次分析的大分子含量，8个迄今未知的韧皮部mRNA的cDNA扩增片段长度多态性。大麦韧皮部分泌物中的转录物与代谢、信号传导和病原体防御相关，例如编码蛋白激酶和病原体和昆虫响应WIR1A（小麦诱导的抗性1A）样蛋白。此外，一维凝胶电泳和随后的部分测序质谱法导致了7个主要蛋白质的鉴定与推定的功能，在应激反应和运输的mRNA，蛋白质和糖。两个发现的蛋白质可能代表一种新的韧皮部移动蔗糖转运蛋白的亚型。值得注意的是，双向电泳证实，有超过250个韧皮部蛋白等待在未来的研究中鉴定。
Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 μl of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies.
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影响因子：
6.9
作者：
Kehr, Julia;Buhtz, Anja
通讯作者：
Buhtz, Anja
影响因子：
4.3
作者：
Gaupels, Frank;Knauer, Torsten;van Bel, Aart J. E.
通讯作者：
van Bel, Aart J. E.
影响因子：
7.2
作者：
Bachem, CWB;vanderHoeven, RS;Visser, RGF
通讯作者：
Visser, RGF
影响因子：
9.2
作者：
Jaeger, Katja E.;Wigge, Philip A.
通讯作者：
Wigge, Philip A.
影响因子：
4.3
作者：
FISHER, DB;FRAME, JM
通讯作者：
FRAME, JM