| Expression, Purification and in vitro Enzyme Activity Assay of Plant Derived GTPase
|
|
Author:
Date:
2015-11-20
[Abstract] Based on gene expression data after biotic stress, the GTPase RabA4c has been suggested to regulate pathogen-induced callose biosynthesis in the model organism Arabidopsis thaliana. We studied the function of RabA4c in its native and dominant negative (dn) isoform. In planta, RabA4c overexpression prevented penetration of the virulent powdery mildew Golovinomyces cichoracearum into epidermal leaf cells. This penetration resistance was caused by enhanced callose deposition at sites of attempted fungal penetration at early time points of infection. By contrast, RabA4c (dn) overexpression did not increase callose deposition or penetration resistance.
In this protocol, we describe the expression, purification and ...
[摘要] 基于生物胁迫后的基因表达数据，已经建议GTP酶RabA4c 在模拟生物拟南芥中调节病原体诱导的胼lose质生物合成。我们研究了其天然和显性负性（dn）同种型中的RabA4c 的功能。 ，RabA4c 过表达阻止了毒性白粉病菌向鸡冠状叶细胞的侵入。这种穿透阻力是由于在感染早期试图真菌穿透的部位处的胼cal质沉积增加引起的。相反，RabA4c（dn）过表达不增加胼cal质沉积或穿透抗性。在本方案中，我们描述了异源表达的GTPase RabA4c的表达，纯化和活性测定从 thaliana ，基于出版物Ellinger 等人（2014）。我们将< em> RabA4c</em>与荧光团mCitrine融合，并在酵母菌株巴斯德毕赤酵母GS115中表达该蛋白质。为了纯化RabA4c，我们使用特异性结合GFP衍生物如mCitrine的GFP-Trap_A 试剂盒（Chromo Tek）。通过使用来自Innova Biosciences的 GTPase测定试剂盒进行酶活性测定。一般来说，我们遵循制造商的指示。
|
|
|
| Measurement of Extracellular Ca2+ Influx and Intracellular H+ Efflux in Response to Glycerol and PEG6000 Treatments
|
|
Author:
Date:
2013-09-20
[Abstract] The characteristics of Ca2+ and H+ fluxes may reflect the activities of aquaporins, as the up-regulation of aquaporin activities is directly associated with the decrease in cytoplasmic H+ concentration and increase in cytoplasmic Ca2+ concentration. The higher aquaporin activities can protect cells against osmotic stresses by altering water flow into and out of the cells. In order to confirm the contribution of aquaporins to the cell tolerance to different osmotic stresses, net Ca2+ and H+ fluxes are measured using the noninvasive micro-test technique (NMT). NMT provides the real-time in situ detection of net ion transport across membranes. Here, we describe the protocol of in situ detection of net Ca2+ ...
[摘要] Ca 2+和H sup +通量的特征可反映水通道蛋白的活性，因为水通道蛋白活性的上调与细胞质H的减少直接相关> + 浓度和细胞质Ca 2+浓度的增加。较高的水通道蛋白活性可以通过改变进入和离开细胞的水流来保护细胞免受渗透压力。为了证实水通道蛋白对细胞对不同渗透胁迫的耐受性，使用非侵入性微测试技术（NMT）测量净Ca 2+和H + +通量， ）。 NMT提供了跨膜的净离子迁移的实时原位检测。在这里，我们描述了原位检测穿过转化的巴斯德毕赤氏酵母的净Ca 2+和 + 通量的方案。 >细胞响应于甘油和聚乙二醇6000（PEG6000）处理。将转化的酵母细胞加载到在聚-L-赖氨酸溶液（0.1％w/v水溶液）中预处理的盖片上。细胞固定后，微电极位于单层细胞群上方。在由计算机操纵的两个偏移点处测量微伏电压差。使用ASET 2.0和iFluxes 1.0软件，微伏电压差可以转换为离子通量。该方法有望促进NMT在微生物学中的应用。我们非常感谢Younger USA（徐越北京）NMT服务中心对稿件的批判性阅读。
|
|
|