Lledías Fernando, Hernández Felipe, Rivas Viridiana, García-Mendoza Abisaí, Cassab Gladys I, Nieto-Sotelo Jorge
Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad # 2001, Col. Chamilpa C.P., 62210, Cuernavaca, Morelos, Mexico.
Jardín Botánico, Instituto de Biología, Universidad Nacional Autónoma de México, Tercer Circuito Exterior, s/n, Coyoacán, Cd. Universitaria, 04510, Ciudad de México, Mexico.
Protein J. 2017 Aug;36(4):308-321. doi: 10.1007/s10930-017-9720-3.
Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-β-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.
景天酸代谢植物具有一些形态特征，如肉质且变小的叶子、厚厚的角质层和下陷的气孔，这些有助于它们防止过度失水和辐射。作为对干旱环境这些形态适应的分子成分，多肉植物除了产生一组未表征的蛋白酶外，还会产生一系列特定化合物，如复合多糖、色素、蜡和萜类化合物等。由于所有这些化合物都会干扰通过电泳技术对蛋白质的分析，从这些来源制备高质量样品是一项真正的挑战。缺乏合适的蛋白质提取方案限制了对这类植物在分子水平上的研究。在此，我们提出了一种快速且可靠的方案，该方案可在1小时内完成，并适用于广泛的植物，且结果具有可重复性。我们能够在龙舌兰亚科（龙舌兰属、丝兰属、万年兰属和巨麻属）、假叶树亚科（酒瓶兰属和酒瓶树属）以及仙人掌科不同成员的提取物中获得分辨率良好的SDS/PAGE蛋白质图谱。该方法基于污染物和蛋白质在丙酮和pH值改变的溶液存在下的不同溶解度。我们推测了皂苷和高分子量碳水化合物在产生适合电泳的样品中的作用。对基本方案的修改允许通过二维电泳（2DE）对样品进行蛋白质组学分析。通过质谱分析和对来自弱刺龙舌兰样品的一个2DE斑点进行从头测序，成功鉴定出呋甾烷醇糖苷26 - O - β - 葡萄糖苷酶（一种参与甾体皂苷合成的酶）。
