An approach for vacuole isolation and vacuolar protein identification by proteomics in apple

Apple, one of the most common fruits consumed worldwide, has a high economic value. Vacuoles are membrane-bound organelles in plant cells that perform many important functions. An increasing number of studies have reported a variety of proteins in a vacuole, with crucial functions in the transport, storage and metabolism of energy and substances. Here, we successfully extracted the vacuoles and vacuolar proteins from the ‘Orin’ apple fruit calli. We collected intact vacuoles with a density of 100/mm2 by mild osmotic lysis and Ficoll density gradient centrifugation. The result of SDS-PAGE showed that lots of vacuolar proteins were isolated and enriched from vacuole samples, and western blotting showed that the purity of the vacuolar protein was high. Furthermore, we showed GO enrichment and KEGG enrichment of isolated vacuolar proteins and selected 41 vacuolar proteins with known functions and 10 vacuolar proteins with unknown functions for functional annotation from the mass spectrometry analysis. This approach for vacuole isolation and vacuolar protein identification will provide a theoretical basis for further study of the function of vacuoles and vacuolar proteins.苹果作为全球最常见的食用水果之一，具备极高的经济价值。液泡（vacuole）是植物细胞内由膜包被的细胞器，可执行诸多重要生理功能。越来越多的研究表明，液泡内存在多种蛋白质，这些蛋白质在能量与物质的转运、储存及代谢过程中发挥关键作用。本研究成功从‘奥瑞恩’（Orin）苹果果实愈伤组织中分离得到液泡及其所含的液泡蛋白：通过温和渗透裂解与菲科（Ficoll）密度梯度离心法，我们获得了密度为100/mm²的完整液泡。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结果显示，从液泡样本中分离并富集得到大量液泡蛋白；蛋白质免疫印迹（western blotting）实验证实，所获液泡蛋白纯度较高。此外，本研究对分离得到的液泡蛋白开展了基因本体（Gene Ontology, GO）富集分析与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析，并通过质谱分析筛选出41个功能已知的液泡蛋白与10个功能未知的液泡蛋白进行功能注释。该液泡分离与液泡蛋白鉴定方法，可为后续液泡及液泡蛋白的功能研究提供理论基础。