Schröter Yvonne, Steiner Sebastian, Weisheit Wolfram, Mittag Maria, Pfannschmidt Thomas
Lehrstuhl für Pflanzenphysiologie, Institut für Allgemeine Botanik und Pflanzenphysiologie, Friedrich-Schiller-Universität Jena Jena, Germany.
Lehrstuhl für Pflanzenphysiologie, Institut für Allgemeine Botanik und Pflanzenphysiologie, Friedrich-Schiller-Universität Jena Jena, Germany ; KWS SAAT AG Einbeck, Germany.
Front Plant Sci. 2014 Oct 29;5:557. doi: 10.3389/fpls.2014.00557. eCollection 2014.
Plant cotyledons are a tissue that is particularly active in plastid gene expression in order to develop functional chloroplasts from pro-plastids, the plastid precursor stage in plant embryos. Cotyledons, therefore, represent a material being ideal for the study of composition, function and regulation of protein complexes involved in plastid gene expression. Here, we present a pilot study that uses heparin-Sepharose and phospho-cellulose chromatography in combination with isoelectric focussing and denaturing SDS gel electrophoresis (two-dimensional gel electrophoresis) for investigating the nucleic acids binding sub-proteome of mustard chloroplasts purified from cotyledons. We describe the technical requirements for a highly resolved biochemical purification of several hundreds of protein spots obtained from such samples. Subsequent mass spectrometry of peptides isolated out of cut spots that had been treated with trypsin identified 58 different proteins within 180 distinct spots. Our analyses indicate a high enrichment of proteins involved in transcription and translation and, in addition, the presence of massive post-translational modification of this plastid protein sub-fraction. The study provides an extended catalog of plastid proteins from mustard being involved in gene expression and its regulation and describes a suitable purification strategy for further analysis of low abundant gene expression related proteins.
植物子叶是一种在质体基因表达方面特别活跃的组织，目的是从原质体（植物胚胎中的质体前体阶段）发育出功能正常的叶绿体。因此，子叶是研究参与质体基因表达的蛋白质复合体的组成、功能和调控的理想材料。在此，我们展示了一项初步研究，该研究使用肝素-琼脂糖和磷酸纤维素色谱法，并结合等电聚焦和变性SDS凝胶电泳（二维凝胶电泳），来研究从子叶中纯化得到的芥菜叶绿体的核酸结合亚蛋白质组。我们描述了从这类样品中高度分辨地生化纯化数百个蛋白质点的技术要求。随后，对用胰蛋白酶处理过的切下的斑点中分离出的肽段进行质谱分析，在180个不同的斑点中鉴定出58种不同的蛋白质。我们的分析表明，参与转录和翻译的蛋白质高度富集，此外，该质体蛋白质亚组分存在大量的翻译后修饰。这项研究提供了一份参与基因表达及其调控的芥菜质体蛋白质的扩展目录，并描述了一种合适的纯化策略，用于进一步分析低丰度的基因表达相关蛋白质。
