Uwase Grace, Enrico Taylor P, Chelimo David S, Keyser Benjamin R, Johnson Russell R
Department of Biology, Colby College.
Department of Biology, Colby College;
J Vis Exp. 2018 Mar 30(133):56728. doi: 10.3791/56728.
The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (β-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.
大麦籽粒的糊粉层是植物中激素调节基因表达的重要模型系统。在糊粉层细胞中，萌发或幼苗早期发育所需的基因由赤霉素（GA）激活，而与应激反应相关的基因由脱落酸（ABA）激活。GA和ABA信号传导机制可通过粒子轰击将报告基因构建体导入糊粉层细胞来进行研究，并使用酶分析法测量由此产生的瞬时表达。本文报道了一种改进的方案，该方案部分自动化并简化了籽粒匀浆步骤和酶分析，与现有方法相比，通量显著提高。使用自动组织匀浆器对籽粒样品进行匀浆，并使用96孔板系统进行GUS（β-葡萄糖醛酸酶）分析。使用该方案的代表性结果表明，磷脂酶D活性可能通过转录因子TaABF1在ABA激活HVA1基因表达中发挥重要作用。
