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Author:
Date:
2016-01-20
[Abstract] Damage to plant organs through both biotic and abiotic injury is very common in nature. Arabidopsis thaliana 5-day-old (5-do) seedlings represent an excellent system in which to study plant responses to mechanical wounding, both at the site of the damage and in distal unharmed tissues. Seedlings of wild type, transgenic or mutant lines subjected to single or repetitive cotyledon wounding can be used to quantify morphological alterations (e.g., root length, Gasperini et al., 2015), analyze the dynamics of reporter genes in vivo (Larrieu et al., 2015; Gasperini et al., 2015), follow transcriptional changes by quantitative RT-PCR (Acosta et al., 2013; Gasperini et al., 2015) or examine additional aspects of the wound ...
[摘要] 通过生物和非生物损伤对植物器官的损害在自然界中是非常常见的。拟南芥（Arabidopsis thaliana）5天龄（5-do）幼苗代表了一种优异的系统，其中在损伤部位和远端无害组织中研究植物对机械创伤的反应。经受单次或重复子叶伤害的野生型，转基因或突变品系的幼苗可用于定量形态学改变（例如根长度，Gasperini等人，2015），分析体内报道基因的动力学（Larrieu等人，2015; Gasperini等人，2015），遵循定量的转录变化RT-PCR（Acosta等人，2013; Gasperini等人，2015）或通过大量下游程序检查伤口反应的其它方面。在这里我们说明如何快速，可靠地卷绕年轻幼苗的子叶，并显示两个启动子驱动表达的β-葡萄糖醛酸酶（GUS）在整个幼苗和主根根分生组织，单个或重复子叶伤害后的行为，分别。我们描述了可以容易地适应具体实验需要的两个程序。
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Author:
Date:
2014-01-05
[Abstract] Determining the localization of proteins within living cells may be very essential for understanding their biological function. Usually for analysis of subcellular localization, a construct encoding the translational fusion of a cDNA of interest with a fluorescent protein (FP) is engineered, transiently expressed in plant cells and examined with confocal microscopy.
In co-localization and interaction studies, two plasmids, each encoding one of the potential interacting/binding partners tagged with an appropriate pair of fluorescence proteins (for instance CFP/YFP) are co-expressed in plant cells. If proteins co-localize in certain cellular compartments it does not necessarily mean that they bind/interact to each other, therefore an additional technique should be applied for in ...
[摘要] 确定蛋白质在活细胞内的定位可能是非常必要的了解其生物学功能。通常对于亚细胞定位的分析，编码感兴趣的cDNA与荧光蛋白（FP）的翻译融合的构建体被改造，在植物细胞中瞬时表达并用共聚焦显微镜检查。在共定位和相互作用研究，在植物细胞中共表达两种质粒，每种质粒编码用适当的荧光蛋白对（例如CFP/YFP）标记的一种潜在的相互作用/结合伴侣。如果蛋白质共定位在某些细胞区室中，其不一定意味着它们彼此结合/相互作用，因此应当应用另外的技术用于体内假定相互作用的验证，/em>荧光寿命成像（FLIM）以检测荧光共振能量转移（FRET）。该协议详细描述了用于验证细菌效应物HopQ1和14-3-3a宿主之间的相互作用的方法蛋白质，并且另外检查HopQ1中的规范14-3-3结合位点中的中心丝氨酸的必要性（Giska等人，2013）。
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